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The synthetic peptide T11F (TCRVDHRGLTF), with sequence identical to a fragment of the constant region of human IgM, and most of its alanine-substituted derivatives proved to possess a significant candidacidal activity in vitro. In this study, the therapeutic efficacy of T11F, D5A, the derivative most active in vitro, and F11A, characterized by a different conformation, was investigated in Galleria mellonella larvae infected with Candida albicans . A single injection of F11A and D5A derivatives, in contrast with T11F, led to a significant increase in survival of larvae injected with a lethal inoculum of C. albicans cells, in comparison with infected animals treated with saline. Peptide modulation of host immunity upon C. albicans infection was determined by hemocyte analysis and larval histology, highlighting a different immune stimulation by the studied peptides. F11A, particularly, was the most active in eliciting nodule formation, melanization and fat body activation, leading to a better control of yeast infection. Overall, the obtained data suggest a double role for F11A, able to simultaneously target the fungus and the host immune system, resulting in a more efficient pathogen clearance.

The synthetic peptide T11F (TCRVDHRGLTF), derived from the constant region of human IgM antibodies, proved to exert a significant activity in vitro against yeast strains, including multidrug resistant isolates. Alanine substitution of positively charged residues led to a decrease in candidacidal activity. A more dramatic reduction in activity resulted from cysteine replacement. Here, we investigated the conformational properties of T11F and its alanine-substituted derivatives by circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy. Peptide interaction with Candida albicans cells was studied by confocal and scanning electron microscopy. T11F and most of its derivatives exhibited CD spectra with a negative band around 200 nm and a weaker positive band around 218 nm suggesting, together with NMR coupling constants, the presence of a polyproline II (PPII) helix, a conformational motif involved in a number of biological functions. Analysis of CD spectra revealed a critical role for phenylalanine in preserving the PPII helix. In fact, only the F11A derivative presented a random coil conformation. Interestingly, the loss of secondary structure influenced the rate of killing, which turned out to be significantly reduced. Overall, the obtained results suggest that the PPII conformation contributes in characterising the cell penetrating and fungicidal properties of the investigated peptides., Competing Interests: The authors declare no conflict of interest.

Background/aims: New antivirals are needed to supplement or replace currently used drugs. The aim of this study was to evaluate the antiviral activity of synthetic peptides derived from physiological proteins., Methods: Vero cell monolayers were infected with herpes simplex virus 1, vesicular stomatitis virus, adenovirus, and coxsackievirus B5 strains in the presence of different concentrations of the selected peptides and viral yield was determined by plaque reduction assays to evaluate the antiviral activity of the peptides. Virucidal activity was evaluated by determining the residual infectivity of viral suspensions treated for 1 h with the peptides at the same concentrations as in the viral yield assays., Results: Among the investigated peptides, the killer peptide proved to exert a considerable antiviral activity against herpes simplex virus, attributable to a direct effect on virus particles, while its derivative K10S showed to be effective against the four investigated virus strains only at the highest concentration tested, yet, the inhibitory effects were only partial., Conclusion: Overall, initial evidence is provided on the antiviral activity of several peptides, as well as of their derivatives. Further investigation is warranted to ascertain the mechanism of action in order to develop new potential antiviral drugs., (© 2019 S. Karger AG, Basel.)

An engineered killer peptide (KP) based on a recombinant anti-idiotypic antibody representing the functional image of a yeast killer toxin (KT) was demonstrated to mediate antimicrobial effects against fungi and viruses. KP binds to murine dendritic cells and macrophages and up-regulate co-receptor expression, thus sustaining CD4+ lymphocyte activation. No immunological data are available in domestic animals thus KP-induced immunomodulation was evaluated in porcine monocyte and lymphocyte subsets. PBMC from healthy adult pigs were stimulated with KP or a scramble peptide (SP), or kept unstimulated for 24, 48 and 72h, and subsequently analyzed by flow cytometry. In monocytes, KP induced a strong dose-dependent shift from a major fraction of CD172α+CD14+ low cells to a predominant fraction of CD172α+CD14+ high cells, known to sustain leukocyte activation/differentiation and inflammatory responses. The CD16+ cell percentages, specifically the CD3+CD16+ natural killer T (NKT) cell fraction and CD16 expression showed an intense and stable dose-dependent increase while the CD3-CD16+ NK cell fraction decreased. CD4+ and CD8+ T cells increased and CD8α and CD8β expression were up-regulated. CD8β+ cytotoxic T cells and CD16+ cells comparably increased. A marked stimulation of activated CD16+CD25+ and CD8β+CD25+ cells was observed at 24h. The increase of CD8α+ cells and CD8α expression were due to increased CD4+CD8α+ (memory T helper) cells, also showing a CD8α+ high phenotype. Concomitantly, the CD4+CD8α- T helper lymphocyte fraction significantly decreased. Overall, KP induced a wide modulation of innate immune and T cells that can exert regulatory and cytotoxic functions, which are fundamental for an efficient Th1 response., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

In recent years, the increase of invasive fungal infections and the emergence of antifungal resistance stressed the need for new antifungal drugs. Peptides have shown to be good candidates for the development of alternative antimicrobial agents through high-throughput screening, and subsequent optimization according to a rational approach. This review presents a brief overview on antifungal natural peptides of different sources (animals, plants, micro-organisms), peptide fragments derived by proteolytic cleavage of precursor physiological proteins (cryptides), synthetic unnatural peptides and peptide derivatives. Antifungal peptides are schematically reported based on their structure, antifungal spectrum and reported effects. Natural or synthetic peptides and their modified derivatives may represent the basis for new compounds active against fungal infections.

Synthetic peptides encompassing sequences related to the complementarity-determining regions of antibodies or derived from their constant region (Fc peptides) were proven to exert differential antimicrobial, antiviral, antitumor, and/or immunomodulatory activitiesin vitroand/orin vivo, regardless of the specificity and isotype of the parental antibody. Alanine substitution derivatives of these peptides exhibited unaltered, increased, or decreased candidacidal activitiesin vitro The bioactive IgG-derived Fc N10K peptide (NQVSLTCLVK) spontaneously self-assembles, a feature previously recognized as relevant for the therapeutic activity of another antibody-derived peptide. We evaluated the contribution of each residue to the peptide self-assembling capability by circular-dichroism spectroscopy. The interaction of the N10K peptide and its derivatives withCandida albicanscells was studied by confocal, transmission, and scanning electron microscopy. The apoptosis and autophagy induction profiles in yeast cells treated with the peptides were evaluated by flow cytometry, and the therapeutic efficacy against candidal infection was studied in aGalleria mellonellamodel. Overall, the results indicate a critical role for some residues in the self-assembly process and a correlation of that capability with the candidacidal activities of the peptidesin vitroand their therapeutic effectsin vivo., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Synthetic peptides, representative of sequences related to the complementarity determining regions and constant region of antibodies, proved to exert in vitro, ex vivo and/or in vivo antimicrobial, antiviral, anti-tumour and/or immunomodulatory activities, conceivably mediated by different mechanisms of action and regardless of the specificity and isotype of the belonging immunoglobulin. Antibody-derived peptides can show intrinsic properties of self-aggregation in β structures, able to assemble on molecular targets and dissociate spontaneously, leading to the formation of hydrogels. Whilst the self-assembled state may provide protection against proteases and the slow kinetic of dissociation assures a release of the active form over time, the receptor affinity is responsible for targeted delivery. Peptides derived from single amino acid substitution of bioactive antibody fragments, adopted as surrogates of natural point mutations, displayed further differential biological activities. Overall, these observations allow to envisage that antibodies could represent an unlimited source of new anti-infective and anti-tumour peptides., (Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.)

This review focuses on antibodies (Abs) and their function in immune protection, with particular emphasis on microbicidal Abs. Some aspects of Abs and Ab-drug conjugates as targeting therapeutic agents are also discussed. The main aim, however, is devoted to Ab-derived peptides modulating functions of the immune system and to the latest experimental evidence of Abs as a source of anti-infective and antitumor peptides derived from their complementarity determining regions and constant regions.

A mouse anti-anti-anti-idiotypic (Id) IgM monoclonal antibody (mAb K20, Ab4), functionally mimicking a Wyckerhamomyces anomalus (Pichia anomala) killer toxin (KT) characterized by fungicidal activity against yeasts presenting specific cell wall receptors (KTR) mainly constituted by β-1,3-glucan, was produced from animals presenting anti-KT Abs (Ab3) following immunization with a rat IgM anti-Id KT-like mAb (mAb K10, Ab2). MAb K10 was produced by immunization with a KT-neutralizing mAb (mAb KT4, Ab1) bearing the internal image of KTR. MAb K20, likewise mAb K10, proved to be fungicidal in vitro against KT-sensitive Candida albicans cells, an activity neutralized by mAb KT4, and was capable of binding to β-1,3-glucan. MAb K20 and mAb K10 competed with each other and with KT for binding to C. albicans KTR. MAb K20 was used to identify peptide mimics of KTR by the selection of phage clones from random peptide phage display libraries. Using this strategy, four peptides (TK 1-4) were selected and used as immunogen in mice in the form of either keyhole limpet hemocyanin (KLH) conjugates or peptide-encoding minigenes. Peptide and DNA immunization could induce serum Abs characterized by candidacidal activity, which was inhibited by laminarin, a soluble β-1,3-glucan, but not by pustulan, a β-1,6-glucan. These findings show that the idiotypic cascade can not only overcome the barrier of animal species but also the nature of immunogens and the type of technology adopted.

A proline-rich peptide of 2733Da, isolated from pig parotid granule preparations was tested against different pathogenic fungi. It showed interesting antifungal activity towards a clinical isolate of Cryptococcus neoformans, with an EC(50) of 2.2μM. Neither cytotoxic nor haemolytic effects were observed towards mammalian cells. Circular dichroism and infrared spectroscopic studies showed that the peptide adopted a combination of polyproline type-II, β-turn and unordered conformations at physiological temperatures. Temperature dependent experiments evidenced a tendency to adopt a polyproline-II helix conformation. From experiments with lipid vesicles, Neutral Red Uptake (NRU), haemolytic assays, and confocal microscopy studies, it could be hypothesized that the peptide may exert its antifungal effect by interacting with an intracellular target rather than through membrane damage., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Synthetic peptides with sequences identical to fragments of the constant region of different classes (IgG, IgM, IgA) of antibodies (Fc-peptides) exerted a fungicidal activity in vitro against pathogenic yeasts, such as Candida albicans, Candida glabrata, Cryptococcus neoformans, and Malassezia furfur, including caspofungin and triazole resistant strains. Alanine-substituted derivatives of fungicidal Fc-peptides, tested to evaluate the critical role of each residue, displayed unaltered, increased or decreased candidacidal activity in vitro. An Fc-peptide, included in all human IgGs, displayed a therapeutic effect against experimental mucosal and systemic candidiasis in mouse models. It is intriguing to hypothesize that some Fc-peptides may influence the antifungal immune response and constitute the basis for devising new antifungal agents.

Antimicrobial peptides represent a promising alternative to traditional drugs in relation to cost, toxicity, and, primarily, the growing problem of drug resistance. Here, we report on the activity against HSV-1 and HSV-2 of a previously described wide-spectrum synthetic decapeptide, Killer Peptide (KP). As determined by plaque reduction assays, treatment with KP at 100 μg/mL resulted in a reduction in the viral yield titer of 3.5 Logs for HSV-1 and 4.1 Logs for HSV-2. Further evaluation of KP antiviral activity focused on the early stages of the virus replicative cycle, including the determination of the residual infectivity of viral suspensions treated with KP. A direct effect of the peptide on viral particles impairing virus absorption and penetration was shown. The toxicity profile proved to be extremely good, with a selectivity index of 29.6 for HSV-1 and 156 for HSV-2. KP was also active against acyclovir (ACV)-resistant HSV isolates, while HSV subcultures in the presence of sub-inhibitory doses of KP did not lead to the emergence of resistant strains. Finally, the antiviral action of KP proved to be synergistic with that of ACV. Overall, these results demonstrate that KP could represent an interesting addition/alternative to acyclovir for antiviral treatment. [ABSTRACT FROM AUTHOR]

Bacterial resistance to β-lactam antibiotics, particularly new generation cephalosporins, is a major public health concern. In Escherichia coli, resistance to these antibiotics is mainly mediated by extended-spectrum β-lactamases (ESBL), which complicates a range of health-threatening infections. These infections may also be biofilm-related, making them more difficult to treat because of the higher tolerance to conventional antibiotics and the host immune response. In this study, we tested as potential new drug candidates against biofilm-forming ESBL-producing E. coli four antimicrobial peptides previously shown to have antifungal properties. The peptides proved to be active in vitro at micromolar concentrations against both sensitive and ESBL-producing E. coli strains, effectively killing planktonic cells and inhibiting biofilm formation. Quantitative fluorescence intensity analysis of three-dimensional reconstructed confocal laser scanning microscopy (CLSM) images of mature biofilm treated with the most active peptide showed significant eradication and a reduction in viable bacteria, while scanning electron microscopy (SEM) revealed gross morphological alterations in treated bacteria. The screening of the investigated peptides for antibacterial and antibiofilm activity led to the selection of a leading candidate to be further studied for developing new antimicrobial drugs as an alternative treatment against microbial infections, primarily associated with biofilms. [ABSTRACT FROM AUTHOR]

Antimicrobial resistance is a major public health concern worldwide. Albeit to a lesser extent than bacteria, fungi are also becoming increasingly resistant to antifungal drugs. Moreover, due to the small number of antifungal classes, therapy options are limited, complicating the clinical management of mycoses. In this view, antimicrobial peptides (AMPs) are a potential alternative to conventional drugs. Among these, Proline-rich antimicrobial peptides (PrAMPs), almost exclusively of animal origins, are of particular interest due to their peculiar mode of action. In this study, a search for new arginine- and proline-rich peptides from plants has been carried out with a bioinformatic approach by sequence alignment and antimicrobial prediction tools. Two peptide candidates were tested against planktonic cells and biofilms of Candida albicans and Candida glabrata strains, including resistant isolates. These peptides showed similar potent activity, with half-maximal effective concentration values in the micromolar range. In addition, some structural and functional features, revealing peculiar mechanistic behaviors, were investigated. [ABSTRACT FROM AUTHOR]

It has been previously demonstrated that synthetic antibody-derived peptides could exert a significant activity in vitro, ex vivo, and/or in vivo against microorganisms and viruses, as well as immunomodulatory effects through the activation of immune cells. Based on the sequence of previously described antibody-derived peptides with recognized antifungal activity, an in silico analysis was conducted to identify novel antifungal candidates. The present study analyzed the candidacidal and structural properties of in silico designed peptides (ISDPs) derived by amino acid substitutions of the parent peptide KKVTMTCSAS. ISDPs proved to be more active in vitro than the parent peptide and all proved to be therapeutic in Galleria mellonella candidal infection, without showing toxic effects on mammalian cells. ISDPs were studied by circular dichroism spectroscopy, demonstrating different structural organization. These results allowed to validate a consensus sequence for the parent peptide KKVTMTCSAS that may be useful in the development of novel antimicrobial molecules. [ABSTRACT FROM AUTHOR]

Background: A peptide of 2,733 Da named SP-E, previously isolated from pig saliva and already described for its antifungal activity and absence of toxicity against mammalian cells, is characterized by a high content of proline residues (70% of entire sequence), that confer structural features probably related to peptide activity. Purpose: The aim of this study was to evaluate the activity of SP-E against Gram-negative bacteria, including drug-resistant clinical isolates. Methods: SP-E and shorter fragments of the same peptide were tested in vitro against the selected bacteria by colony forming unit assays. Scanning electron microscopy and confocal microscopy were also applied. SP-E potential therapeutic activity was evaluated in vivo in a Galleria mellonella model of bacterial infection. Results: SP-E proved to be active against the tested bacteria with EC50 values in the micromolar range. Though maintaining antibacterial properties, the shorter peptides showed lower activity in respect to the parental molecule. Kinetics of killing action and nonmembranolytic internalization within Escherichia coli and Pseudomonas aeruginosa cells strongly suggested a cytosolic mechanism of action involving one or more intracellular molecular targets. A single injection of SP-E exerted a therapeutic effect in G. mellonella larvae infected with P. aeruginosa. Conclusion: The biological properties of SP-E strongly back this peptide as a new promising multitasking antimicrobial molecule. [ABSTRACT FROM AUTHOR]

The killer peptide KP is a synthetic decapeptide derived from the sequence of the variable region of a recombinant yeast killer toxin-like microbicidal single-chain antibody. KP proved to exert significant activities against diverse microbial and viral pathogens through different mechanisms of action, but little is known of its effect on apicomplexan protozoa. The aim of the present study was to evaluate the in vitro activity of KP against Toxoplasma gondii, a globally widespread protozoan parasite of great medical interest. The effect of KP treatment and its potential mechanism of action on T. gondii were evaluated by various methods, including light microscopy, quantitative PCR, flow cytometry, confocal microscopy, and transmission electron microscopy. In the presence of KP, the number of T. gondii tachyzoites able to invade Vero cells and the parasite intracellular proliferation were significantly reduced. Morphological observation and analysis of apoptotic markers suggested that KP is able to trigger an apoptosislike cell death in T. gondii. Overall, our results indicate that KP could be a promising candidate for the development of new anti-Toxoplasma drugs with a novel mechanism of action. [ABSTRACT FROM AUTHOR]

Antibodies (Abs) are emerging as an important class of therapeutic agents for the treatment of various human diseases, often conjugated to drugs or toxic substances. In recent years, the incidence of cancer and infectious diseases has increased dramatically, making it imperative to discover new effective therapeutic molecules. Among these, small peptides are arousing great interest. Synthetic peptides, representative of variable and constant region fragments of Abs, were proved to exert in vitro, ex vivo and/or in vivo anti-microbial, anti-viral, anti-tumour and/or immunomodulatory activities, mediated by different mechanisms of action and regardless of the specificity and isotype of the Ab. Some of these synthetic peptides possess the ability to spontaneously and reversibly self-assemble in an organised network of fibril-like structure. Ab fragments may represent a novel model of targeted anti-infective and anti-tumour auto-delivering drugs. [ABSTRACT FROM AUTHOR]

We previously reported that a synthetic peptide with sequence identical to a CDR of a mouse monoclonal antibody specific for difucosyl human blood group A exerted an immunomodulatory activity on murine macrophages. It was therapeutic against systemic candidiasis without possessing direct candidacidal properties. Here we demonstrate that a selected peptide, N10K, putatively deriving from the enzymatic cleavage of the constant region (Fc) of human IgG1, is able to induce IL-6 secretion and pIkB-α activation. More importantly, it causes an up-regulation of Dectin-1 expression. This leads to an increased activation of b-glucan-induced pSyk, CARD9 and pIkB-α, and an increase in the production of pro-inflammatory cytokines such as IL-6, IL-12, IL-1β and TNF-α. The increased activation of this pathway coincides with an augmented phagocytosis of non opsonized Candida albicans cells by monocytes. The findings suggest that some Fc-peptides, potentially deriving from the proteolysis of immunoglobulins, may cause an unexpected immunoregulation in a way reminiscent of innate immunity molecules. [ABSTRACT FROM AUTHOR]

Background: It has been documented that, independently from the specificity of the native antibody (Ab) for a given antigen (Ag), complementarity determining regions (CDR)-related peptides may display differential antimicrobial, antiviral and antitumor activities. Methodology/Principal Findings: In this study we demonstrate that a synthetic peptide with sequence identical to VHCDR3 of a mouse monoclonal Ab (mAb) specific for difucosyl human blood group A is easily taken up by macrophages with subsequent stimulation of: i) proinflammatory cytokine production; ii) PI3K-Akt pathway and iii) TLR-4 expression. Significantly, VHCDR3 exerts therapeutic effect against systemic candidiasis without possessing direct candidacidal properties. Conclusions/Significance: These results open a new scenario about the possibility that, beyond the half life of immunoglobulins, Ab fragments may effectively influence the antiinfective cellular immune response in a way reminiscent of regulatory peptides of innate immunity. [ABSTRACT FROM AUTHOR]

Mycoses still represent relevant opportunistic infections worldwide, although overshadowed in recent years by other severe and more widespread infections. Moreover, deep-seated mycoses are often accompanied by unacceptably high mortality rates. Etiologic agents include endogenous components of the mycobiota, Candida and Malassezia species above all, and exogenous species, both yeasts and filamentous fungi. Old and new fungal pathogens are increasingly characterized by resistance to the existing antifungal agents, making imperative the search for effective and safe new therapeutics. Among the candidate molecules proposed in recent decades, synthetic peptides derived from the complementarity determining and constant regions of diverse antibodies (Abs), as well as the translated products of Ab-encoding genes, have proved of considerable interest. Their anti-infective activities, regardless of the specificity and isotype of the originating Ab, will be briefly presented and discussed in the light of their different mechanisms of action. Intriguing suggestions on the possible function of Abs after their half-life will be presented, following the recent detection, in human serum, of an antimicrobial Ab-derived peptide. Overall, Abs could represent a source of biologically active, highly flexible peptides, devoid of detectable toxicity, which can be easily synthesized and manipulated to be used, alone or in association with already available drugs, for new anti-infective strategies. [ABSTRACT FROM AUTHOR]