Effect Of PKA Regulatory Subunits On Autophagy Of Aspergillus Fumigatus

Objective: Aspergillus fumigatus(A.fumigatus),an opportunistic pathogen,is a common airborne filamentous fungus,often found in high-temperature compost and rotting plants.The perturbation of its living environment will release a large number of conidia,which cause life-threatening invasive aspergillosis(IA)in people with low immunity.Autophagy,as a programmed cell death,is significantly different from apoptosis.Autophagic cell death hasa prominent feature in morphology asmassive autophagic vacuolization of the cytoplasm.Autophagy is widely studied in yeast and mammalian cells.Yeast is a single celled organism.Mammalian is a complex advanced multicellular organism.Filamentous fungi,as a low-level multicellular organism,are not studied much in the field of autophagy,but they have attracted more and more attention.The autophagy studies of A.fumigatus showed: EDTA could induce autophagy of A.fumigatus,including the formation of vacuoles and the production of autophagic particles;atg1 gene knockout resulted in the disappearance of mycelial autophagic particles and the loss of mycelial extension capability in nutrient deficiency medium.Under starvation conditions,ATG8 accumulated within the vacuoles in WT,but ATG8 failed to accumulate in the vacuoles of the atg1 gene knockout mutant under starvation conditions.In fungus,the c AMP signaling pathway is responsible for regulating various processes and has been shown to be critical for the virulence of immunosuppressive invasive pulmonary aspergillosis in murine models.The main regulator of the c AMP pathway is protein kinase A(PKA).Inactive PKA is a tetramer consisting of 2 regulatory subunits(pka R)and 2 catalytic subunits(pka C),which passes through the c AMP and subsequent components.PKA is an important signaling molecule that regulates growth,metabolism,and proliferation in cells.Deletion of the regulatory subunit of PKA in A.fumigates caused decreased survival rate with time,and accumulated massive vacuoles in the cytoplasm.This paper preliminarily explored the relationship between PKA and autophagy in A.fumigatus,and the study of autophagy and cell death mechanism will provide the theoretical basis for the control of A.fumigates infection for clinical treatment.Method: The autophagy level were detected in wild-type(WT)and gene-deleted mutant(?pka R)strain.The conidia of WT and ?pka R were placed in sterile H2 O for 1-7 days,survival rates was compared,and survival rates at different times were plotted.Single colonies of WT and ?pka R were inoculated on nutrient-rich medium(YG)and nutrient-deficient medium(WA),and the diameters of colonies were measured daily to determine the differences in the extension ability of their mycelia.Morphological changes were observed on the two strains cultured for a certain period of time,after autophagy induction with EDTA and addition of roteinase inhibitor PMSF,their mycelia and vacuolar autophagosomes were observed by DIC microscope.Dansylcadaverine(MDC)staining was performed on autophagosomes in mycelia of WT and ?pka R,and observed under a confocal laser scanning fluorescent microscope.Real-time PCR was used to analyze the m RNA expression levels of autophagy-associated genes such as Afatg1,Afatg8,and Afatg11,and quantitatively analyzed the relationship between PKA and autophagy.The mice were infected with ?pka R and Western blot was used to detect the expression of autophagy-related proteins in mouse lung alveolar macrophages.Results: Compared with the WT strain,the survival rate of the ?pka R strain after incubating in sterile water for 7 days significantly decreased,indicating that the deletion of pka R reduces the survival rate of A.fumigates condia.After incubating on WA solid media for same period of time,the diameters of single colonies were measured and results showed that the diameters of colonies of ?pka R were significantly decreased,indicating that deletion of pka R gene reduced the extension ability of A.fumigatus hyphae.DIC microscopy showed that the number of autophagic vacuoles of ?pka R strain increased significantly,after autophagy induction with EDTA and addition of PMSF,the number of autophagic bodies of ?pka R strain was higher than that of WT,indicating that knockout of pka R can promote autophagy of A.fumigatus.MDC staining of autophagosome,oberserved under confocal laser scanning microscope,showed that the number of fluorescent paticles of ?pka R strain was significantly higher than that of WT strain.After addition of EDTA,the number of autophagosomes of ?pka R strain was significantly higher than that of WT.This suggests that the knockout of pka R leads to a stronger autophagic response.Real-Time PCR results showed that the m RNA expression levels of autophagy-related genes Afatg1,Afatg8 and Afatg11 in ?pka R mutant were significantly higher than those of WT strain.Western blot results showed that the expression of LC3-II protein produced by lung alveolar macrophages increased significantly in ?pka R condia infected mice.Conclusion: 1.Knockout of PKA regulatory subunits can promote autophagy in A.fumigatus.2.The PKA regulatory subunit may increase the autophagy level of A.fumigatus by promoting the expression of Afatg1,Afatg8 and Afatg11.3.The knockout of pka R can reduce spore survival and mycelial elongation.