Identification And Functional Study Of A Pathogenicity Related Gene FRT1 In Cryptococcus Neoformans

Cryptococcus neoformans is a encapsled basidiomycetous fungus that is widely existed in nature.C.neoformans is an opportunistic fungal pathogen that primarily infects immunocompromised people to cause cryptococcal pneumonia and meningitis.According to statistics,C.neoformans can infect about one million people,resulting in over 600,000 deaths 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.So far,given the limited drugs to fight Cryptococcosis and the continual emergence of drug-resistant strains,the difficulty in the treatment of C.neoformans infection has increased.C.neoformans has caused great threat to human health and safety because of its high mortality.C.neoformans has two mating types,?and a,that can mate to form a dikaryotic hyphae and produce basidiospores.C.neoformans has three major virulence factors,namely,polysaccharide capsule,melanin and growing at 37 degrees Celsius.In addition,C.neoformans also has other virulence factors,such as its superoxide dismutase and mannitol.C.neoformans has also become a model organism for the study of fungal genetics and pathogenicity,and its important signaling pathways related to sexual reproduction and virulence have also been extensively studied.In this study,a virulence pathogenicity-related gene FRT1 was identified based on previous studies.The function of FRT1 was studied based on bioinformatics analysis and molecular biology approaches,which includs the spatio-temporal expression of FRT1,construction of FRT1 knockout,complement and overexpression strains,and the effect of FRT1 on the growth and pathogenicity in C.neoformans.This study primarily clarified the role of FRT1 in the regulation of morphological development and pathogenicity in C.neoformans.The main results are as follows:1.Sequence analysis of FRT1A functionally unknown gene was isolated from the LC-MS datas and named FRT1.Sequence analysis revealed that the coding region of the gene is 2215bp in length and contains two very short introns encoding a 72.3KD protein.Domain analysis revealed that Frt1 protein contains three ZnFC₂H₂ zinc finger domains that may regulate transcription and expression of downstream genes.2.Spatial and temporal expression of FRT1To explore the expression pattern of FRT1,we cloned the promoter of FRT1 and fused with mCherry.After expression in C.neoformans,the red fluorescence signal was observed in yeast cell,mating hyphae and basidiospores using confocal microscopy.The results showed that red fluorescence signal could be detected in all development stages of C.neoformans,indicating that FRT1 promoter is a constitutive promoter and regulates expression of FRT1 in various development stages of C.neoformans.We also explored the expression of FRT1 in the early mating stage by qRT-PCR and Western blot.The results showed that the expression level of FRT1 gradually decreased with the prolongation of time in the early mating stage.To explore the subcellular localization of Frt1,we generated the Frt1-mCherry and GFP-Frt1 fusion expression strains which were observed and photographed under confocal fluorescent microscopy.The results showed that Frt1 was localized in the cytoplasm in yeast and mycelium.3.Construction of FRT1 gene knockout,complementation and overexpression strainsTo further study the function of FRT1,the alpha mating type frt1?mutant strain were generated by using Split Marker strategy and homologous recombination.Then we isolated the a mating type frt1?mutant from the spores generated by mating of C.neoformans.The FRT1 gene overexpression strain FRT1^OE was generated by expressing the FTT1 gene under the control of the actin promoter using biolistic transformation.The FRT1 in situ complementary strains were also generated by transformation of the FRT1 in situ complementary vector into the frt1?mutants.4.Phenotypic analysis of frt1?mutant,FRT1 complement and overexpression strainsPhenotypic analysis of the above strains revealed that there was no difference between wild-type strains,frt1?mutants and complementary strains in capsule formation,melanin production and growth at 37?.However,the FRT1 overexpression strain FRT1^OE produced less capsule and showed growth defects at 37?.The frt1?mutants and the FRT1^OE strains are sensitive to SDS,but not to Congo red,indicating that Frt1 may regulates the cell membrane integrity in C.neoformans.Mating assays showed that the frt1?mutants and overexpression strains FRT1^OE can produce mating hyphae,but not produce spores,indicating that Frt1 is also essential for the sexual reproduction in C.neoformas.In order to further explore the mechanism of non-spore production of the frt1?mutant,we constructed strains expressing the Nop1-mCherry fusion protein,a protein localize in the nuclears,and observe the change of the nuclears over time in mating mycelium.The results showed that the two nucleas in the mating hyphae could fuse but coulnd not undergo meiosis in the frt1?mutants,indicating that the deletion of FRT1 may block the process of meiosis.5.Virulence studiesTo test the role of Frt1 in the pathogenesis of C.neoformans,C57BL6 mice were intranasally infected with 10⁵ cells of each yeast strain.The 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 and mice survival curve was worked out.To compare the fungal burdens,lungs,brains and spleens from mice infected by H99,the frt1?mutants and FRT^OE strains were isolated at the end time point and homogenized using a homogenizer in PBS buffer.Lungs,brains and spleens were also fixed in 10%formalin solution,and sent to the company for section preparation.The virulence test showed that the virulence of the frt1?mutant was significantly attenuated and the FRT1^OEE strains are completely avirulent.The fungal burdens of infected mice were measured as yeast CFU per gram fresh organ and the results showed that no yeast cells were recovered in FRT1^OE-infected spleen and brain,which are consistent with the histopathological results of the infected organs.Summarily,a new pathogenicity-related gene FRT1 was identified in C.neoformans in this study.Fuctional analysis showed that Frt1 may regulate the cell membrane integrity,fungal virulence and sexual reproduction in C.neoformans.Functional analysis of Frt1 offered helps to elucidate the mechanism of pathogenicity and sexual reproduction in C.neoformans.