Studies on action mechanism of tenecin 3, an insect antifungal protein, against the pathogenic fungus Candida albicans

Abstract

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 pe...