Autophagy Regulates Sexual Reproduction And Virulence In Cryptococcus Neoformans

Cryptococcus neoformans is a major encapsulated yeast pathogen that often enters human body by inhalation to cause fungal meningoencephalitis in immunocompromised individuals,resulting in over 600,000 fatalities worldwide annually.Due to the prevalence of AIDS,the use of cancer and chemotherapy drugs,and the use of immunosuppressive drugs after transplantation in recent years,the morbidity of Cryptococcosis increase constantly in population.Cryptococcosis has become an common complications of AIDS patients in many countries outside of China,which is also the main cause of death.The incidence of Cryptococcosis in China also shows a trend of increasing year by year and shows a unique tendency of infection on immunocompetent people.For the treatment of Cryptococcosis,the therapeutic effects of fungal antibiotics currently used clinically is not often good.Even if use combination drugs,the total effective rate of treatment is less than 75%,which makes the prevention and control of Cryptococcosis in China face a severe challenge.Proteolysis is a highly specific process that are involved with a series of proteases with diverse backgrounds.There are two major proteolytic pathways in eukaryotic cells:the ubiquitin proteasome system and lysosome-mediated proteolysis(e.g.autophagy).Autophagy is an evolutionarily highly conserved process of protein degradation in eukaryotes.In the process,autophagosomes consisting of a bilayer membrane encapsulates damaged proteins or cell organelles,and transport them to the lysosome or vacuole for degradation,allowing macromolecules to be degraded and reused by cells.Autophagy plays an important role in maintaining cell homeostasis,regulating cell differentiation and development,and determining cells longevity.There is a certain extent of similarity between autophagic protein degradation and ubiquitinated protein degradation.For example,autophagy-related proteins Atg8 and Atg12 play an ubiquitin-like role in the autophagy pathway,known as ubiquitin-like proteins.In this study,we focus on the ubiquitin-like proteins Atg8,Atgl2 and its interacting protein Atg5,which are involved in autophagic protein degradation pathways.The role of the above autophagic proteins in fungal development and virulence are analyzed by using methods such as gene knock-out,complementation and overexpression,localization of fluorescent labeling proteins,phenotypic analysis and so on.The main results were as follows:First,we identified the autophagy-related proteins Atg5,Atg8 and Atg12 in C.neoformans by homologous sequence alignment using the Saccharomyces cerevisiae Atg5,Atg8 and Atg12 sequences.Using the technology of in vitro homologous recombination,we constructed autophagy-related protein fusion with green fluorescent protein,Atg5-EGFP,Atg8-EGFP and Atgl2-EGFP expression vectors.The green fluorescent strains expressing Atg5-EGFP,Atg8-EGFP and Atgl2-EGFP fusion protein in H99 background were obtained by biolistic tansformation and drug resistence selection,respectively.Subcellular localization analysis showed that the localization of the three autophagy proteins in the cells was basically consistent with the model organism S.cerevisiae.Secondly,we constructed the gene knockout vectors of ATG5,ATG8 and ATG12.The Aatg5,?atg8 and ?atgl2 mutants in which the ATG5,ATG8 and ATG12 genes were knocked out respectively were obtained by using biolistic transformation and in vivo homologous recombination.Meanwhile,the gene complementation vecrors for Aatg5,Aatg8 and Aatgl2 mutants were constructed and corresponding gene-complementing strains were obtained after biolistic transformation and drug resistence screening.We studied the phenotypes of the above strains under different stress conditons such as high temperature(37?),high osmotic pressure(NaCl/KCl),oxidative stress(H2O2),high detergent activity(SDS)and the production of melanin and so on.The Aatg5,Aatg8 and ?atg12 strains did not show any phenotypic difference when compare with the wild type strain H99 undre the above stress conditions.In the nitrogen starvation induction experiment,cells grown to logarithmic growth phase were starved culturing in SD-N medium for more than 4 hours with the protease inhibitor PMSF.Microscopic observations revealed that there were a large amount of autophagosomes aggregated in the vacuole of the wild-type strain,while no autophagosome was found in the yeast cells of Aatg5,Aatg8,and ?atg12 mutant strains,indicating that the autophagic process were blocked in the mutants.Then the survival rate of the mutant strains after starvation was explored.The wild type,mutants and gene complemented strains were inoculated into SD-N medium at a concentration of 2x107 cells/ml,respectively.After induction in the starvation medium for 0 h,24 h,48 h,72 h and 96 h,the cell cultures were spread on SDA medium and CFUs were counted on the plate and drawn the survival curves of cells,respectively.The survival results showed that the mutant strains were more sensitive to nitrogen starvation and less viable.C.neoforrrmans is basidiomycetes having two mating types of a and a.They can mate and produce basidiospores when induced on MS medium.In this study,the a mating type mutant strains were obtained by mating using the a mating type mutants and KN99a strain.The mating ability of the Aatg5,Aatg8 and Aatg12 mutant strains were also analyzed through the bilateral mating experiment.The Aatg5,Aatg8 and Aatg12 mutants could mate and form dicaryon hypha on MS medium,but had no basidiospores,indicating that the sexual reproduction process were blocked in the mutant strains.To further explore why the mutant strains did not produce spores after mating,the nuclear localization gene NOP1 was selected to construct the fusion expression vector Nopl-GFP and Nop1-mCherry and transformed into the wild type strains H99 and KN99a and mutant strains ?atg5,?atg8,?atg12,respectively by biolistic transformation.The fluorescent labled strains with nuclear localization were selected and used for mating assays.After 14 days induction in dark at 25? on MS media,the mating hyphae were examined using a confocal fluorescence microscopy.As a result,it was found that the two nuclei in the wild-type strain were able to fuse normally to perform meiosis and produce spores,while in the mutants stains ?atg5,?atg8,and ?atg12,the nucleus can undergo fusion,but cannot pass through the meiosis process,resulting in no basidiospore formation on the basidia of the mutant strains.Finally,the pathogenicity of ?atg5,?atg8 and ?atg12 mutant strains were tested in C57BL6 mouse.Groups of 10 female eight-week-old C57BL6 mice were intranasally infected with 105 cells of each yeast strain.The body weight of mice are recorded everyday 2 weeks after inoculation.Over the course of the experiments,animals that appeared moribund or in pain were sacrificed by CO2 inhalation.Survival data from the murine experiments were statistically analyzed between paired groups using the logrank test with PRISM version 7.0.To compare the fungal burdens,lungs,brains and spleens from mice infected by H99,the Aatg mutants,or the complemented strain of the Aatg mutants were isolated at the end time point,fixed in 10%formalin solution,and sent to the company for section preparation.Lungs,brains and spleens were also isolated and homogenized using a homogenizer in PBS buffer.Resuspensions were diluted,100 ?L of each dilution was spread on YPD medium with ampicillin and chlorampHenicol,and colonies were determined after 2 days of incubation at 30?.The results showed,compared with the wild-type strain,the pathogenicity of the?atg5 mutant was slightly reduced,and the ?and ?atg12 mutants was significantly reduced.In summary,3 autophagy-related genes ATG5,ATG8,and ATG12 were we identified and analyzed in C.neoformans in this study.Subcellular localization of Atgs were also analyzed.The gene knock-out mutants ?atg5,?atg8,and ?atg12 were obtained by biolistic transformation and phenotypes of the mutant strains were also tested under different stress conditions.Autopahgy were blocked in the above mutants strains which were sensitive and less viable under nitrogen starvation.The mutant strains could not produce spores after mating and the likely reason is that the nucleus of the mutant strains were not able to undergo meiosis after fusion.Virulence studies showed that the pathogenicity of ?atg5 strain was slightly reduced,while that of ?atg8 and ?atg12 mutants were significantly reduced.