Antimicribial And Antitumor Activity Of Trichokonins From Trichoderma Koningii SMF2

The peptaibols are a family of antimicrobial peptides isolated from soil fungi that exhibit anti-bacterial and anti-fungal properties. Peptaibols are synthesized by non-ribosomal peptide synthetases, which contain high proportions ofα-aminoisobytyric acid (Aib) and adoptα-helical structure. The N-terminal residues of these peptides are usually acetylated or rarely acetylated, and an amino alcohol is linked by a peptide bond at the C-terminal end. The presence of the Aib residues in the sequence promotes the formation of the helical structures due to the steric constraints imposed by the second methyl group on the Cαatom. All peptaibol structures determined to date are highly helical. The imino acids Pro or Hyp tend to promote formation of bends or kinks in these structures. Peptaibols are amphipathic in nature and this property allows many of them to form voltage-dependent ion channels in lipid bilayer membranes. Studies were focused on the structures and interaction with the model membranes to date. Only crystal structures of monomeric forms have been determined thus far. Based on which, models have been developed for multimeric transmembrane channels. But the antimicrobial properties and antitumor properties of peptaibols have not been studied thoroughly. Up to now, 317 peptaibols have been sequenced and 190 of which produced by Trichoderma. Trichoderma spp. are free-living fungi that are highly interactive in root, soil and foliar environments. They have been shown to act, and are commercially applied as biological control agents (BCAs) against fungal pathogens. In our screening work of biological control agents, Trichoderma koningii SMF2 could produce heat resistant secondary metabolites against many phytopathogenic fungal strains. In this study, these metabolites were identified and their antimicrobial and antitumor activities were studied.I . Isolation, purification and identification of Trichokonins Purification of these metabolites was achieved by combination of gel filtration and high performance liquid chromatography. Being identified by liquid chromatography electrospray ionisation tandem mass spectrometry, the active metabolites proved to be three known peptaibols: Trichokonin VI, VII and VIII (Huang et al., 1995; Landreau et al., 2002), named kangningmeisu.II. Antimicrobial property of Trichokonin VIAntibacterial activities of antimicrobial metabolites produced by Trichoderma koningii SMF2 were bactericidal against all Gram-positive bacteria tested including medically important Staphylococcus aureus (SA) and multi-drug resistant S. aureus (MDRSA). They exhibited antimicrobial activity against a range of Gram-positive bacterial and fungal phytopathogens also. Trichokonins showed moderate antimicrobial activities against all the Gram-positive bacteria tested, Staphylococcus aurues (MIC = 25μM), Bacillus substilis (MIC = 25μM). However, the MIC against Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa) could not be determined even at 200μM. Time-killing curves indicate that the multi-drug resistant strain showed a higher survival rate in the presence of 25μM Trichokonins. When inoculated for 20 times in 25μM Trichokonins, the sensitivity of both strains to Trichokonins decreased greatly, and the MICs were increased above 100μM both. These results indicated that both strains became resistant to Trichokonins after the treatment. The mechanism of the resistance requires further experimentation.III. Detection of antitumor activities of Trichokonin VIThree tumor cell lines (A549, HepG2, BGC823) were used for the detection of antitumor activities of Trichokonin VI. MTT assay indicated that Trichokonin VI greatly inhibited the growth of these three tumor cells and the IC50 were about 25μM. Fluorescence microscopy revealed different morphological changes of cells when treated with Trichokonin VI, especially the condensation of nucleus and vacuolization of the cytoplasm. All these results suggested that Trichokonin VI was cytotoxic against tumor cells, and it maybe a potential anti-tumor drug candidate.IV. Antitumor mechanism involved in Trichokonin VI (TK VI) in HepG2 cellsBased on MTT assay, TK VI led to significant inhibition in proliferation of HepG2 cells in a dose-dependent manner. Indeed, our results provided strong evidence that the TK VI-mediated cell death of HepG2 cells displayed some characteristics of paraptosis (i.e., massive cell death, mitochondrial disruption, the appearance of empty vacuoles and external membrane integrity). Characteristically, cell death triggered by TK VI was accompanied by degradation of DNA large fraction, loss of the mitochondrial transmembrane potential. Moreover, TK VI changed the permeability of HepG2 cells, which might be due to its formation of Ca²⁺ permeable pores in the cell membrane causing Ca²⁺ influx into cells. Furthermore, western blot analysis showed down-regulation of Bcl-xL. TK VI also triggered a cleavage of caspase-9 precursors into activated derivatives, a process inhibited by benzyloxycarbonyl-Val-Ala-Asp-(OMe) fluoromethyl ketone (z-VAD-fmk). Subsequently, cellular endogenous poly (ADP-ribose) polymerase (PARP) protein was significantly activated. Altogether, these data suggested that TK VI-induced cell death defined a novel form of paraptosis in a caspase-dependent manner, connecting the Ca²⁺ involved pathway.