PkaR Gene Impacts The Apoptosis Of The Germinate Spores Of Aspergillus Fumigatus

Objective Aspergillus fumigatus is a kind of opportunistic pathogenic fungus,human body using alveolar macrophages to kill the spores of A.fumigatus.A.fumigatus is the most common causative agent of invasive aspergillosis in immunocompromised patients.Limited antifungal therapies are available due to high toxicity,low efficacy rates,and growing drug resistance.Activation of endogenous apoptosis reactions is a promising approach to combat invasive aspergillosis and other fungal diseases.Better understanding of these pathways might provide the basis for the development of novel anti-fungal therapeutics against aspergillosis.Protein kinase A takes part in regulating growth,morphology and toxicity of fungus.Protein kinase A is a regulator of respond to stress in eukaryote,pkaR is the regulator of Protein kinase A.Human body depends on oxidative stress to kill the germinate spores of A.fumigatus.Thus it is very important to study the mechanism of pkaR gene in regulating the apoptosis of germinate spores of A.fumigatus.Method Compare apoptosis parameters of the germinate spores of wild type and pkaR gene deleted mutant after undergo oxidative stress to study whether pkaR gene gets involved in regulating apoptosis in A.fumigatus.Cultivate the spores of wild type and mutant into germinated spores,using hydrogen peroxide as oxidative stress and incubate with germinated spores.After the incubation exam the viability of germinate spores by methylene blue staining and recultivation;detect ROS production by cultivating with DCFH-DA;measure DNA fragmentation by TUNEL method;judge integrate of cytomembrane by PI staining.Results The result of methylene blue staining shows that after expose to oxidative stress the viability of pkaR gene deleted mutant is higher than wild type;the examination of ROS production shows that wild type has more ROS production under low oxidative stress but less ROS production under high oxidative stress,on the contrary pkaR gene deleted mutant has less ROS production under low oxidative stress but more ROS production under high oxidative stress,there is significant difference of ROS production between the mutant and wild type,compare with wild type;TUNEL staining indicates that as the increasing of the intensity of oxidative pressure the amount of apoptosis cells of wild type undergos rising and falling but there is no significant change of the apoptosis of mutant after enhancing oxidative stress;PI staining shows under oxidative stress the cytomembrane of wild type germinated spores got damaged more than mutant indicates that there are more cells undergo cell death,mutant has stronger ability to resist oxidative damage.Conclusion Methylene blue staining as a method to exam the viability of fungus can apply to estimate the viability of germinate spores of A.fumigatus;the germinate spores of the pkaR gene deleted mutant has a higher survival rate and become less sensitive to oxidative stress indicates that pkaR gene impact the resistance to oxidative stress of germinate spores of A.fumigatus;there is significant difference of ROS production between wild type and mutant after undergo oxidative stress shows pkaR gene takes part in regulating ROS production of germinate spores of A.fumigatus;there is a big difference of the amount of apoptosis cells between wild type and mutant under the same level of oxidative stress reveals that pkaR gene takes part in regulating the process of apoptosis of A.fumigatus.