Mechanism Underlying Conidial Death In Aspergillus Flavus Induced By Thymol

Aspergillus flavus is a well-known saprotrophic and pathogenic fungus for its colonization of cereal grains,legumes and tree nuts,and many strains can produce toxic compounds,especially aflatoxin,which leads to rapid death and chronic outcomes such as hepatocellular carcinoma.To eliminate the adverse effects of A.flavus,some antifungal agents(e.g.voriconazole)have been developed to treat infections caused by this fungus.However,recent reports have showed clinical failures due to the acquirement of resistance to azoles by A.flavus.Deep perception of mechanisms underlying death of A.flavus will contribute to the development of new efficient drugs against this pathogen.Thymol is one of major chemical constituents of essential oil in Thymus vulgaris.Many studies suggested that thymol was capable to inhibit the growth of bacteria,fungi and yeast.In this study,we found that thymol could induce A.flavus spores lysis and death.To further understand the mechanism underlying conidial death,we managed to provide insights into the type of conidial death by thymol and identify its target in A.flavus associated with its lethal effects.The main results were listed as follows:1.Fungicidal effects of thymol on A.flavus sporesThymol showed efficient antifungal activity against A.flavus.The growth of spores was almost completely inhibited in the presence of more than 80?g/m L thymol.Through viability assay,more than99.9% cells were killed efficiently by thymol.It was also supported by the results from the scanning electron microscopy(SEM)results,indicating that considerable amounts of spores were lysed.ROS and NO,were often considered to be signal molecules associated with cell death.Large amounts of ROS and NO were also generated when the spores of A.flavus were exposed to thymol.The removal of either ROS or NO attenuated lysis and death of spores exposed to thymol.Especially,ROS depletion also blocked the generation of NO.Taken together,ROS was involved in spore death induced by thymol via the induction of NO.2.Conidial apoptosis occurring in response to thymol treatmentIn general,apoptosis and necrosis were included in the most death modes in eukaryotes.Here we observed conidial apoptosis occurring 3h post thymol treatment with PI and Hoechst 33342 double fluorescent staining through fluorescent microscopy and flow cytometer.And the percentage of apoptotic conidia increased along the treatment,and most of conidia were in apoptosis 6h post treatment.By using TUNNEL staining another apoptosis features was identified 9h post treatment i.e.DNA breakage injury.Further study indicated that conidial mitochondria membrane potential was losing in response to thymol with JC-1 fluorescent dye,which was the marker of occurring mitochondria membrane damage.It is well accepted that mitochondria membrane damage can lead to the release of cytochrome C(cyt C)from mitochondria to cytosol,which activates caspase 9 initializing apoptosis.With the kit,we detected the caspase 9 activity in conidia dramatically increasing post the treatment of thymol,and the addition of Z-VAD-FMK(a universal caspase inhibitor)was able to block increasing caspase 9 activity and attenuate conidial apoptosis.This indicated that thymol led to conidial apoptosis via the induction of mitochondrial damage.3.Voltage-gated K+ channel involving the initialization of conidial apoptosisThe potential target of thymol leading to conidial apoptosis in A.flavus was investigated by prediction based on the database of protein-drug interaction for human(DRAR-CPI).Voltage-gated K+channel protein(PDB id: 1ZSX)in human was identified as one of the potential target of thymol,and is high homologous to KCNAB in A.flavus.Voltage-gated K+ channel is often mentioned to be responsible for apoptosis.Further 3D-molecular mode of KCNAB was structured using I-TASSER and predicated the interaction between thymol and KCNAB by docking.The results showed the interaction mode between thymol and KCNAB was very similar to that between thymol and 1ZSX.With the fluorescent probe of K+(PBFI),we determined a transient eruption of conidial K+ level in A.flavus in response to the addition of thymol.The addition of a inhibitor(4-Ap)special for voltage-gated K+ channel eliminated this stimulation on K+ level by thymol and simultaneously decreased the amounts of apoptotic conidia.These results strongly supported the previous prediction: thymol targeted KNCAB,which promoted conidial apoptosis in A.flavus.4.Construction of KCNAB-knockout mutantSince KCNAB played a vital role in the conidial apoptosis by thymol,we wondered what would happen to a KCNAB-deletion mutant.We managed to construct a KCNAB-knockout mutant in CA14?pyr G ku70.Consequent results showed deletion of KCNAB led to disordered conidial germination,i.e.vigorous germination in distilled waters and weak germination in culture medium.Further study showed the ability of ?KCNAB invading onion and peanut was significantly lower than the control.This was probably due to lack of ?subunit(KCNAB)led to voltage-gated K+ channel over-sensitive to extrinsic stimulation.Exposure of ?KCNAB to thymol led to more violent K+ eruption and more conidial apoptosis comparing to wild strain.These results not only supported our hypothesis on overacting of K+ channel,but also our prediction that voltage-gated K+ channel KCNAB involving conidial apoptosis by thymol.Taken together,a pathway leading to conidial apoptosis in A.flavus by thymol was elucidated that thymol stimulated conidial K+ eruption,which led to the mitochondrial damage,ROS generation,activation of caspase and DNA breakage.Our study provided a novel target “KCNAB” with great potential to be applied in the development of new drugs against aspergillosis and strategy against aflatoxin contamination.