Tenecin 3 is a glycine-rich antifungal protein of 78 residues isolated from the hemolymph of insect Tenebrio molitor larva. Tenecin 3 was initially screened as a candidacidal protein in the hemolymph that did not inhibit the growth of Gram positive and Gram negative bacteria. The characteristic of the amino acid sequence and the antifungal specificity of tenecin 3 have raised a lot of interests for the mechanism of its antifungal action. However, the antifungal mechanism has not yet been studied due to its very low availability from natural source. To overcome this problem, bacterial expression systems were used to obtain the recombinant tenecin 3 proteins. The pET and pRSET expression vector systems provided rapid and convenient methods to generate the recombinant tenecin 3 proteins in E. coli. His-tagged fusion proteins were purified by $Ni^{2+}$-chelating affinity column chromatography. The recombinant tenecin 3 protein without any fusion was also purified by $Ni^{2+}$-chelating affinity column chromatography by taking advantage of the high histidine content of tenecin 3. These recombinant proteins all showed the same characteristics of natural tenecin 3; no antibacterial activity, yeast-type specific antifungal activity, salt-dependent antifungal activity, and non-hemolytic activity. The large quantities of the recombinant tenecin 3 proteins were easily obtained by using this expression and purification system.
As an initial step in understanding the antifungal mechanism of tenecin 3, I also examined how tenecin 3 interacts with the pathogenic fungus Candida albicans to exert its antifungal action in comparison with a pore-forming mechanism, the most prevalent mechanism of antimicrobial proteins. The serial truncation experiment showed that two domains at N terminal half and C terminal half regions are sufficient for the antifungal activity. Tenecin 3 did not induce the release of a fluorescent dye trapped in the artificial membrane vesicles. It did not perturb the membrane potential of C. albicans during the initial interaction, either. These results suggest that tenecin 3 does not disrupt the lipid membrane through direct interaction such as pore-formation. Fluorescence confocal microscopy and FACScan analysis revealed that tenecin 3 is rapidly translocated into the cytoplasmic space by energy-dependent and temperature-dependent manners. This internalization is also dependent on the ionic environment and cellular metabolic (growth) state. These results suggest that internalization of tenecin 3 into the cytoplasm of C. albicans is mediated by a fungal endocytic uptake. The internalized tenecin 3 was dispersed in the cytoplasm, and the loss of the cell-viability occurred after internalization of tenecin 3. The internalized tenecin 3 induced the release of an anionic fungal vacuole specific dye, DFFDA from the vacuole, while the vacuolar membrane seems to be intact. The fact that the characteristics of tenecin 3 internalization and its effect differ from those of other known antifungal proteins suggests that tenecin 3 exerts its antifungal action through a novel mechanism.
