Internalization of tenecin 3 by a fungal cellular process is essential for its fungicidal effect onCandida albicans

Abstract

Tenecin 3 is a glycine-rich, antifungal protein of 78 residues isolated from the insect Tenebrio molitor larva. As an initial step towards understanding the antifungal mechanism of tenecin 3, we examined how this protein interacts with the pathogenic fungus Candida albicans to exert its antifungal action. Tenecin 3 did not induce the release of a fluorescent dye trapped in the artificial membrane vesicles and it did not perturb the membrane potential of C. albicans by the initial interaction. Fluorescence confocal microscopy and flow cytometric analysis revealed that tenecin 3 is rapidly internalized into the cytoplasmic space in energy-dependent and temperature-dependent manners. This internalization is also dependent on the ionic environment and cellular metabolic states. These results suggest that the internalization of tenecin 3 into the cytoplasm of C. albicans is mediated by a fungal cellular process. The internalized tenecin 3 is dispersed in the cytoplasm, and the loss of cell viability occurs after this internalization. [14]. These studies have shown that the formation of transmembrane pores or ion channels on the cellular membranes cause the leakage of essential metabolites. The disruption of the microbial cell structure is the major killing mechanism for these cationic, antimicrobial proteins. This mechanism is also generally applicable to the antifungal action mechanism of these proteins, and even to the antiviral mechanism [15]. Other well known antifungal mechanisms of plant antifungal proteins, such as chitinases [16] [19], include the disruption of the cell wall structure and interference in cell wall synthesis. However, some antimicrobial proteins, including the cysteine-rich plant defensin Rs-AFP 2 [20], the insect antifungal protein drosomycin [5], and the glycine-rich insect antifungal protein AFP [21], show no growth inhibition for Gram negative and Gram positive bacteria. It has been proposed that the channel formation through direct protein-lipid interaction is not a probable antifungal mechanism for these proteins although their action mechanisms have not yet been fully elucidated [22]. There is a high sequence homology between Rs-AFP 2 and drosomycin, while AFP has no significant homology with these proteins [21]. We previously isolated tenecin 3 as an antifungal protein from the insect Tenebrio molitor and found that it had no antibacterial activity [23]. As tenecin 3 shares 37.3% identity with AFP from Sarcophaga peregrina, it can be categorized into an antifungal protein group with AFP and another insect antifungal protein, holotricin 3, from Holotricia diomphalia [24]. Tenecin 3 is also a glycine-rich protein, as are AFP and holotricin 3. However, little is known about the action mechanism of AFP and its homologs. The binding of AFP to Candia albicans was thought to be the cause of some damage to the cells even though this binding is weak and dependent on ionic environments [21]. Tenecin 3 consists of 78 amino acids and has distinctive features in its primary Abbreviations: AFP, ant...

Modulation of In Vitro Fungicidal Activity of Human Lactoferrin againstCandida albicansby Extracellular Cation Concentration and Target Cell Metabolic Activity

“…Figure 5 shows the results of a representative experiment performed under candidacidal conditions in which cell suspensions in 5 mM PPB (pH 7.4) were exposed to hLf (5 M) for 2 h. A similar result was obtained in control assays with cells treated with amphotericin B (Fig. 5), an antifungal agent that causes depolarization of C. albicans cell membranes (22).…”

Mechanisms of Antimicrobial Peptide Action and Resistance

“…For example, the glycine-rich antifungal peptide tenacin-3 quickly enters the C. albicans cytoplasm via an energy-dependent mechanism that is influenced by target cell metabolic status and ionic environment (Kim et al, 2001). Yet, this peptide does not appear to induce membrane permeabilization or depolarization in doing so.…”

Peptide Antimicrobial Agents

“…The peptide is able to quickly enter the cytoplasm of C. albicans in an energy-dependent mechanism influenced by the metabolic status of the target cells and the ionic environment (125). It does not result in membrane permeabilization or depolarization (125), although pegylated tenecin-3 peptide can result in K ϩ leakage from C. albicans (146), possibly explaining its improved antifungal activity.…”

“…It does not result in membrane permeabilization or depolarization (125), although pegylated tenecin-3 peptide can result in K ϩ leakage from C. albicans (146), possibly explaining its improved antifungal activity. The antifungal mechanism of native tenecin-3 is unknown, but the loss of cell viability occurs after the peptide enters the host cell cytoplasm (125).…”