The greater wax moth Galleria mellonella has been increasingly used as a model host to determine Candida albicans virulence and efficacy of antifungal treatment. The G. mellonella lysozyme, similarly to its human counterpart, is a member of the c-type family of lysozymes that exhibits antibacterial and antifungal activity. However, in contrast to the relatively well explained bactericidal action, the mechanism of fungistatic and/or fungicidal activity of lysozymes is still not clear. In the present study we provide the direct evidences that the G. mellonella lysozyme binds to the protoplasts as well as to the intact C. albicans cells and decreases the survival rate of both these forms in a time-dependent manner. No enzymatic activity of the lysozyme towards typical chitinase and β-glucanase substrates was detected, indicating that hydrolysis of main fungal cell wall components is not responsible for anti-Candida activity of the lysozyme. On the other hand, pre-treatment of cells with tetraethylammonium, a potassium channel blocker, prevented them from the lysozyme action, suggesting that lysozyme acts by induction of programmed cell death. In fact, the C. albicans cells treated with the lysozyme exhibited typical apoptotic features, i.e. loss of mitochondrial membrane potential, phosphatidylserine exposure in the outer leaflet of the cell membrane, as well as chromatin condensation and DNA fragmentation.
Anionic antimicrobial peptides constitute an integral component of animal innate immunity, however the mechanisms of their antifungal activity are still poorly understood. The action of a unique Galleria mellonella anionic peptide 2 (AP2) against fungal pathogen Candida albicans was examined using different microscopic techniques and Fourier transform infrared (FTIR) spectroscopy. Although the exposure to AP2 decreased the survival rate of C. albicans cells, the viability of protoplasts was not affected, suggesting an important role of the fungal cell wall in the peptide action. Atomic force microscopy showed that the AP2-treated cells became decorated with numerous small clods and exhibited increased adhesion forces. Intensified lomasome formation, vacuolization, and partial distortion of the cell wall was also observed. FTIR spectroscopy suggested AP2 interactions with the cell surface proteins, leading to destabilization of protein secondary structures. Regardless of the anionic character of the whole AP2 molecule, bioinformatics analyses revealed the presence of amphipathic α-helices with exposed positively charged lysine residues. High content of the α-helical structure was confirmed after deconvolution of the IR absorption spectrum and during circular dichroism measurements. Our results indicated that the antimicrobial properties of G. mellonella AP2 rely on the same general characteristics found in cationic defense peptides.
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