Peptides of the Constant Region of Antibodies Display Fungicidal Activity

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

Innate immunity from serum protein derivatives

E’ noto che esiste un ampio gruppo di proteine, incluse proteine della segnalazione endocrina, della matrice extracellulare, della cascata del complemento e del latte, contenenti al loro interno altre unità funzionali, che potrebbero essere rilasciate in vivo in seguito a tagli proteolitici, dotate di attività biologiche non prevedibili a partire dalla sequenza o dall’attività della proteina precursore. L’esistenza di questi frammenti peptidici, denominati criptidi, potrebbe riflettere un meccanismo evolutivo in grado di ampliare la gamma di attività biologiche associate ad una particolare proteina, offrendo interessanti opportunità per la comprensione e la modulazione di vari processi biologici e nuove prospettive per eventuali strategie terapeutiche alternative. In questo scenario, la ricerca oggetto del progetto di Dottorato è stata focalizzata allo studio dell’attività microbicida di frammenti peptidici di anticorpi e altre proteine sieriche, riscontrati in campioni di siero umano. I frammenti peptidici individuati sono derivati dalla regione costante CH3 della catena µ di IgM (K40H), da albumina, frammenti C4 e C3 del complemento e fibrinogeno (K13L, G17K, S17K, D15R e D15T). In particolare, i peptidi K40H e K13L hanno mostrato la più significativa attività fungicida in vitro nei confronti di diversi miceti lievitiformi, inclusi ceppi resistenti a farmaci antifungini. Gli studi effettuati hanno evidenziato per i due peptidi differenti proprietà strutturali e suggerito il coinvolgimento di differenti meccanismi d’azione fungicida. L’efficacia dei peptidi K40H e K13L è stata osservata in un modello ex vivo di infezione da Candida albicans in frammenti di mucosa orale porcina mantenuti in coltura. L’attività terapeutica di K40H e K13L è stata inoltre dimostrata in vivo, in un modello di candidosi sistemica sperimentale in larve del lepidottero Galleria mellonella. Nello stesso modello in vivo, così come nei confronti di cellule di mammifero in coltura e di eritrociti e cellule mononucleate del sangue periferico umane in vitro, è stata verificata l’assenza di tossicità e di attività emolitica e genotossica aspecifica di entrambi i peptidi. Nel complesso, i risultati ottenuti dimostrano che alcuni frammenti peptidici presenti nel siero, derivati da anticorpi e da altre proteine sieriche, analogamente a quanto precedentemente dimostrato per peptidi con sequenza identica a frammenti dalle regioni determinanti la complementarietà e della regione costante di anticorpi, possono esercitare un’attività microbicida. Proteine sieriche, inclusi gli anticorpi, potrebbero rappresentare una fonte illimitata di peptidi a potenzialità anti-infettiva. La dimostrazione della presenza in vivo di frammenti peptidici ad attività fungicida include anticorpi e albumina nel novero delle proteine dalle quali possono derivare frammenti criptici, che già comprendeva proteine della cascata complementare e fibrinogeno, e potrebbe suggerire un loro nuovo ruolo nell’omeostasi antifungina.There is clear evidence that a large group of proteins, including proteins associated with endocrine signalling, the extracellular matrix, the complement cascade and milk, could give rise in vivo, through proteolytic cleavage, to hidden peptides with bioactivities that are often unpredictable from the sequence and activity of the parent protein. The existence of these peptide fragments, designed as cryptides, could reflect an evolutionary mechanism to expand the functionality of proteins, providing additional opportunities for understanding and modulating various biological processes related to a specific protein, and new perspectives for protein-based therapeutic strategies. In this setting, the object of the PhD research project is focused on the study of microbicidal activity of peptide fragments from antibodies and other serum proteins, found in human serum samples. The identified peptide fragments are derived from CH3 constant region of IgM µ chain (K40H), albumin, C4 and C3 complement fragments, and fibrinogen (K13L, G17K, S17K, D15R e D15T). In particular, K40H and K13L peptides showed the most significant fungicidal activity against various yeast isolates in vitro, including strains resistant to common antifungal agents. The research showed different structural properties for these peptides, and the involvement of different mechanisms of fungicidal action was suggested. K40H and K13L were shown to be effective in an ex vivo model of Candida albicans infection using porcine oral mucosa. The in vivo therapeutic activity of K40H and K13L was demonstrated in a model of systemic experimental candidiasis using the larval form of the insect Galleria mellonella. In the same in vivo model, as well as against cultured mammalian cells, human erythrocytes and peripheral blood mononuclear cells, both peptides showed to be devoid of toxicity, haemolytic and genotoxic activity. Overall, the results show that peptide fragments found in the serum, derived from antibodies and other serum proteins, can exert microbicidal activity, similarly to what previously demonstrated for peptides with sequence identical to fragments of the complementarity determining regions and the constant regions of antibodies. Serum proteins, including antibodies, could represent an unlimited source of peptides endowed with anti-infective potential. The demonstration of the presence of fungicidal peptide fragments in vivo include antibodies and albumin in the group of proteins, which already comprised components of the complement cascade and fibrinogen, that could give rise to cryptic fragments and could suggest a new role of these proteins in antifungal homeostasis

Antifungal activity of the C-terminal peptide from human serum albumin

Introduction In recent years, a number of studies showed the biological activities of peptides derived by proteolytic cleavage of physiological proteins. These functional units, called cryptides, can be characterized by activities similar or different to those of the precursor protein. The present study was aimed to evaluate the fungicidal properties of a synthetic peptide (K13L, KKLVAASQAALGL), representing the C-terminal fragment of human albumin found in serum. Materials and Methods The synthetic peptide was obtained by solid-phase synthesis chemistry and evaluated for its biological properties by consolidated experimental methods. The in vitro fungicidal activity was investigated against different yeast species, inclusive of multidrug resistant strains. The ex vivo and in vivo anticandidal efficacy was evaluated in a model of C. albicans infection in porcine oral mucosa and in experimentally infected larvae of Galleria mellonella, respectively. Furthermore, haemolytic, cytotoxic and genotoxic properties were verified. The effect of peptide treatment on C. albicans cells was studied by confocal microscopy and transmission and scanning electron microscopy. Results K13L peptide proved to be fungicidal in vitro against the investigated yeasts at micromolar concentrations, to reduce fungal infiltration in porcine oral mucosa ex vivo, and to exert a therapeutic effect in vivo in the invertebrate animal model, without showing toxic effects on mammalian cells. Microscopic studies demonstrated that the peptide penetrates and accumulates in fungal cells causing gross morphological alterations in cellular structure. Conclusions Overall, our data prove that fragments from physiological serum proteins, as K13L peptide, may exert an antifungal activity, as previously shown for antibody-derived peptides, suggesting a potential role of these molecules in antifungal homeostasis and establishing serum proteins as a source of new antimicrobial agents