F-box Protein Cdc4 Regulates Sexual Reproduction And Virulence In Cryptococcus Neoformans

Cryptococcal meningitis is a kind of fungal disease that seriously endangers human life and health,and cannot be treated effectively.Desiccated yeast cells or basidiospores of C.neoformans or its spores,which are ubiquitous in the environment,can be inhaled through the respiratory tract and infect people with immunodeficiency or low immunity,such as AIDS patients,cancer patients,organ transplant patients or COVID-19 patients.Such people,as a result of the deficiency of immune system,cannot remove the C.neoformans effectively,which caused lung invasion and infection of other organs of the human body with blood circulation.C.neoformans eventually penetrates the blood-brain barrier to infect the central nervous system and causes the deadly cryptococcal meningitis,resulting in hundreds of thousands of deaths each year.Depth analysis of the morphological development and virulence mechanism of C.neoformans is helpful for us to find new drug targets for the treatment of cryptococcosis.The Ubiquitin-proteasome system（UPS）in which F-box protein is involved,specifically degrades most proteins in eukaryotic cells,and the specific degradation of substrate protein is determined by F-box protein.F-box protein is crucial for maintaining the physiological homeostasis of organisms,which not only affects the growth and development of animals and plants,but also is closely related to the research and treatment of human diseases,including cancer,glycolipid disorders,and other diseases.Meanwhile,the F-box proteins also play a significant role in fungal growth,sexual reproduction,virulence and other aspects.In this study,we explored the spatiotemporal expression of F-box protein Cdc4 of C.neoformans and its role in growth,morphological development,stress responses,and pathogenicity.The main results and conclusions are as follows:1.Identification and expression analysis of CDC4we analyzed the gene sequence of CDC4 and found that the full-length coding region of the CDC4 gene is 3126 bp,including three introns and four exons,encoding a protein Cdc4 with a total length of 991 amino acids.The protein domain prediction analysis of Cdc4 showed that it contained 1F-box protein domain and 8 WD40 protein domains,indicating that Cdc4 was a typical f-box protein.Then,we analyzed the spatiotemporal expression of the F-box protein Cdc4 in C.neoformans.In the spatial localization analysis of Cdc4,the fusion expression strain of GFP-Cdc4 was observed by fluorescence microscopy,and it was found that the green fluorescence was localized in the nucleus and cell membrane of the strain of GFP-Cdc4,and was not transferred due to the influence of stress conditions.GFP-Cdc4 strains mating experiment showed that the GFP-Cdc4 fusion protein was located in the nucleus and cell membrane of the yeast cells,hyphae,basidia,and basidiospores of C.neoformans.The results indicate that Cdc4 was located in the nucleus and cell membrane in C.neoformans.We analyzed the expression of CDC4 in the early mating stage and different developmental stages of C.neoformans by qRT-PCR and inducing P_CDC4-mCherry strains mating.The results showed that CDC4 gene was expressed in the yeast cells,mating hyphae,basidiospore and basidia,and the expression of CDC4 in the early mating stage increased at first and then decreased,suggesting the CDC4 may play a role in the mating process of C.neoformans.2.Phenotypic analysis of cdc4Δmutants,CDC4 complementation and overexpression strainsNext,in order to further evaluate the function of Cdc4 in C.neoformans,we generated the cdc4Δmutant strains,CDC4 complementation or overexpression strains to explore the role of Cdc4 in stress responses,sexual reproduction,and pathogenicity of C.neoformans.Classical virulence factor testing found that there was no significant growth difference between wild type,cdc4Δmutant strain,CDC4 complementation or overexpression strains in capsule formation,melanin production,and growth at 37oC,indicating that the deletion of CDC4 gene did not affect the growth of three classical virulence factors of C.neoformans.In the stress responses detection,we found that the CDC4 overexpression strains were sensitive to SDS but not Congo red,indicating that the overexpression of CDC4 would affect the membrane integrity of C.neoformans.Subsequently,we did fungal mating analysis and found that the cdc4Δmutants can produce mating hyphae and basidia,but does not produce basidiospores,indicating that the deletion of CDC4blocked the sporulation of C.neoformans,suggesting that the CDC4 gene played a critical role in the process of sexual reproduction of C.neoformans.Approved for this,we constructed Nop1-mCherry fluorescent strains to further explore why the sporulation of the cdc4Δmutants was blocked.By observing the distribution of red fluorescence located in the nucleus after mating,we found that there were only one or two nuclei in the basidia produced by the mating of cdc4Δmutant strains.Thus,it was speculated that after CDC4 deletion,C.neoformans can only conduct nuclear fusion in the process of mating,but could not conduct nuclear division which led to the block of meiosis,and finally i resulted in the inability to produce basidiospores,thus affecting the sexual reproduction process of C.neoformans.3.Virulence testNext,the inhalation model was used to test the virulence of cdc4Δmutant strains in C57 BL/6mice.The animal study showed that all the mice infected with cdc4Δmutant strains survived on the day of termination of the experiment（80 days after infection）.CFU statistics and the tissue biopsies showed that there was a small amount of C.neoformans cell recovered from the lungs infected by cdc4Δmutant and the alveolar tissue remains intact.No yeast cells were recovered and no damage was found from the brains and spleens of the mice infected by cdc4Δmutant strain.Moreover,to better understand the dynamics of the cdc4Δmutants-host interaction during the infection process,the cdc4Δmutants were used to infect the mice and fungal burdens in infected lungs were examined at different time points.The results showed that with the passage of time,the fungal burden in the lung tissue of the mice infected with wild-type strains is on the rise,and the lung tissue damage is serious;in contrast,the cdc4Δmutant causes little damage in the infected lung and that the fungal burden in the lung remains at a low but persistent level throughout infection.These results showed that the cdc4Δmutant is hypo-virulent in a murine model of systemic infection.In summary,the deletion of the CDC4 gene leads to the loss of post-mating sporulation capacity and pathogenicity of C.neoformans,which indicates that F-box protein Cdc4 plays a crucial role in the sexual reproduction and pathogenicity of C.neoformans.