Anti-infective antibody-derived peptides active against endogenous and exogenous fungi

Mycoses still represent relevant opportunistic infections worldwide, although overshad-owed 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 charac-terized 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

Wickerhamomyces yeast killer toxins’ medical applications

Possible implications and applications of the yeast killer phenomenon in the fight against infectious diseases are reviewed, with particular reference to some wide-spectrum killer toxins (KTs) produced by Wickerhamomyces anomalus and other related species. A perspective on the applications of these KTs in the medical field is provided considering (1) a direct use of killer strains, in particular in the symbiotic control of arthropod-borne diseases; (2) a direct use of KTs as experimental therapeutic agents; (3) the production, through the idiotypic network, of immunological derivatives of KTs and their use as potential anti-infective therapeutics. Studies on immunological derivatives of KTs in the context of vaccine development are also described

Activity of two antimicrobial peptides against Enterococcus faecalis in a model of biofilm-mediated endodontic infection

Enterococcus faecalis is a common cause of biofilm-associated opportunistic infections, which are often difficult to treat. The formation of E. faecalis biofilms on the dentinal walls of the root canal is frequently the cause of endodontic treatment failure and secondary apical periodontitis. In a preliminary work, two recognized antifungal peptides, KP and L18R, showed antibacterial activity against planktonic E. faecalis cells at micromolar concentrations. Moreover, L18R proved to reduce the biomass in the early stage of E. faecalis biofilm development on polystyrene plates, while a qualitative biofilm inhibition was demonstrated on hydroxyapatite disks by confocal laser scanning microscopy (CLSM). The aim of this study was to better characterize the effect of both peptides on E. faecalis biofilm. A reduction in metabolic activity after peptide treatment was detected by Al-amar Blue assay, while a remarkable impairment in the architecture of E. faecalis biofilms on hy-droxyapatite disks, along with a significant reduction in viable bacteria, was caused mostly by L18R, as assessed by CLSM and scanning electron microscopy. The lack of cytotoxicity of the investigated peptides against L929 murine fibroblasts was also determined. Obtained results suggest L18R as a promising candidate for the development of new strategies for endodontic infection control

Antibacterial effects of two synthetic peptides against Enterococcus faecalis biofilms: A preliminary in vitro study

Aim: Current endodontic techniques are unable to fully eradicate intracanal bacteria. Thus, new agents that effectively eliminate endodontic pathogens are needed. The aim of this study was to assess the antibacterial properties of two synthetic peptides, namely KP and L18R, against planktonic cells and biofilms of the endodontic pathogen Enterococcus faecalis. Methodology: KP and L18R bactericidal activity against E. faecalis ATCC 29212 was evaluated by colony forming unit assays and the half maximal effective concentration (EC50) was calculated. The effect of peptides on E. faecalis biofilm formation onto polystyrene plates was also assessed by the crystal violet assay. Confocal laser scanning microscopy (CLSM) analysis was carried out to compare the effects of KP, L18R and a Ca(OH)2 saturated solution in an in vitro model of dental infection consisting in 2-day-old E. faecalis biofilms grown on hydroxyapatite disks. Results: Both KP and L18R showed strong bactericidal activity against planktonic E. faecalis. L18R proved to be 10-folds more ef fective than KP (KP and L18R EC50 values=4.520×10-6 M and 3.624×10-7 M, respectively). Peptides inhibited E. faecalis biofilm formation in a dose-dependent manner and L18R resulted more effectivethan KP. CLSM images showed that Ca(OH)2, KP and L18R remarkably impaired E. faecal is biof i lms pre -grown on hydroxyapatite. Conclusions: KP and L18R effectively inhibited E. faecalis, both in planktonic and biofilm form. L18R demonstrated a more potent antibacterial activity than KP. These preliminary results suggest that antimicrobial peptides may represent a promising new strategy for endodontic infection control

A peptide found in human serum, derived from the c-terminus of albumin, shows antifungal activity in vitro and in vivo

The growing problem of antimicrobial resistance highlights the need for alternative strategies to combat infections. From this perspective, there is a considerable interest in natural molecules obtained from different sources, which are shown to be active against microorganisms, either alone or in association with conventional drugs. In this paper, peptides with the same sequence of fragments, found in human serum, derived from physiological proteins, were evaluated for their antifungal activity. A 13-residue peptide, representing the 597–609 fragment within the albumin C-terminus, was proved to exert a fungicidal activity in vitro against pathogenic yeasts and a therapeutic effect in vivo in the experimental model of candidal infection in Galleria mellonella. Studies by confocal microscopy and transmission and scanning electron microscopy demonstrated that the peptide penetrates and accumulates in Candida albicans cells, causing gross morphological alterations in cellular structure. These findings add albumin to the group of proteins, which already includes hemoglobin and antibodies, that could give rise to cryptic antimicrobial fragments, and could suggest their role in anti-infective homeostasis. The study of bioactive fragments from serum proteins could open interesting perspectives for the development of new antimicrobial molecules derived by natural sources

Therapeutic effect of an antibody-derived peptide in a Galleria mellonella model of systemic candidiasis

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

Isolation of a Wickerhamomyces anomalus yeast strain from the sandfly Phlebotomus perniciosus, displaying the killer phenotype

The yeast Wickerhamomyces anomalus has been studied for its wide biotechnological potential, mainly for applications in the food industry. Different strains of W. anomalus have been isolated from diverse habitats and recently from insects, including mosquitoes of medical importance. This paper reports the isolation and phylogenetic characterization of W. anomalus from laboratory-reared adults and larvae of Phlebotomus perniciosus (Diptera: Psychodidae), a main phlebotomine vector of human and canine leishmaniasis. Of 65 yeast strains isolated from P. perniciosus, 15 strains were identified as W. anomalus; one of these was tested for the killer phenotype and demonstrated inhibitory activity against four yeast sensitive strains, as reported for mosquito-isolated strains. The association between P. perniciosus and W. anomalus deserves further investigation in order to explore the possibility that this yeast may exert inhibitory/killing activity against Leishmania spp

In silico predicted antifungal peptides: In vitro and in vivo anti-candida activity

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

In vitro and in vivo anti-Candida activity and structural analysis of killer peptide (KP)-derivatives

The previously described decapeptide AKVTMTCSAS (killer peptide, KP), derived from the variable region of a recombinant yeast killer toxin-like anti-idiotypic antibody, proved to exert a variety of antimicrobial, antiviral, and immunomodulatory activities. It also showed a peculiar self-assembly ability, likely responsible for the therapeutic effect in animal models of systemic and mucosal candidiasis. The present study analyzed the biological and structural properties of peptides derived from KP by substitution or deletion of the first residue, leaving unchanged the remaining amino acids. The investigated peptides proved to exert differential in vitro and/or in vivo anti-Candida activity without showing toxic effects on mammalian cells. The change of the first residue in KP amino acidic sequence affected the conformation of the resulting peptides in solution, as assessed by circular dichroism spectroscopy. KP-derivatives, except one, were able to induce apoptosis in yeast cells, like KP itself. ROS production and changes in mitochondrial transmembrane potential were also observed. Confocal and transmission electron microscopy studies allowed to establish that selected peptides could penetrate within C. albicans cells and cause gross morphological alterations. Overall, the physical and chemical properties of the first residue were found to be important for peptide conformation, candidacidal activity and possible mechanism of action. Small antimicrobial peptides could be exploited for the development of a new generation of antifungal drugs, given their relative low cost and ease of production as well as the possibility of devising novel delivery systems

Vaccination of Heifers with Anaflatoxin Improves the Reduction of Aflatoxin B-1 Carry Over in Milk of Lactating Dairy Cows

It was previously reported that injection of anaflatoxin B-1 (AnAFB(1)) conjugated to keyhole limpet hemocyanin (KLH), together with Freund's adjuvant, was effective in inducing in cows a long lasting titer of anti-aflatoxin B1 (AFB(1)) antibodies (Abs), cross-reacting with other aflatoxins, which were able to hinder, proportionally to their titer, the secretion of aflatoxin M-1 (AFM(1)) into the milk of cows continuously fed with AFB(1). According to anti-AFB(1) Ab titer, 50% of the vaccinated cows were recognized as high responder animals. In an attempt to prepare a more effective formulation for vaccination of cows, it was compared the immunogenicity, in Holstein Friesian heifers, of AnAFB(1) covalently conjugated to KLH or to recombinant diphtheria toxin (CRM197) molecules, and injected together with various adjuvants. This study demonstrated that injection of AnAFB(1) conjugated to KLH and mixed with complete (priming) and incomplete Freund's adjuvant (boosters), as in the previous schedule of immunization, was the most effective regimen for inducing Ab responses against AFB(1), although pre-calving administration could increase the effectiveness of vaccination, resulting in 100% high responder animals. After one booster dose at the beginning of the milk production cycle, anti-AFB(1) Ab titers were comparable to those recorded at the end of the immunization schedule, and proved to be effective in reducing significantly AFB(1) carry over, as AFM(1), from feed to milk. Pre-calving vaccination of dairy heifers with conjugated AnAFB(1), adjuvated with complete and incomplete Freund's adjuvant, may represent the most effective tool for preventing the public health hazard constituted by milk and cheese contaminated with aflatoxins