Identification And Functional Analysis F-box Protein Fbp1 Downstream Substrate Grp1 Of Cryptococcus Neoformans

Cryptococcus neoformans is an opportunistic yeast fungal pathogen that widely exists in nature and mainly infects immunocompromised individuals such as AIDS and cancer patients.C.neoformans has two mating types:αand a,and has a bipolar mating system.Through sexual reproduction,C.neoformans can switch between single-celled yeast form and multicellular hyphae state.This morphological change helps to infect the host,evade the immune response,and spread.The spores finally formed are also the important survival structures of C.neoformans,which are its transmission vectors and infectious propagules.According to statistics,there are at least 180,000 deaths caused by cryptococcal infection worldwide every year.Although the progress of the current anti-retroviral treatment methods for cryptococcosis has been made progress,the mortality rate caused by cryptococcal infection is still very high,so the treatment of cryptococcosis is still a challenge to be overcome by experts and scholars around the world.It has been reported that the F-box protein Fbp1 in C.neoformans is a part of the SCF E3 ubiquitin ligase complex and is critical for fungal virulence and sexual reproduction.Therefore,identifying the substrates of Fbp1 may be the key to understand how the ubiquitin-proteasome system（UPS）regulates cryptococcal virulence and sexual reproduction.In the previous identification of the potential downstream substrates of F-box protein Fbp1,our research group obtained a Glycine-rich RNA binding protein Grp1 and showed that deletion of the GRP1 gene affected the meiosis of Cryptococcus during the sexual reproduction process,resulting in the failure to produce basidiospores.Therefore,this study aims to clarify the interaction and function between F-box protein Fbp1 and Grp1,and lay a foundation for the study of the mechanism of the ubiquitin-proteasome system regulating cryptococcal pathogenicity and sexual reproduction.By analyzing the domains of Grp1,we found that Grp1 has three different spliceosomes,and the encoded proteins are 16.263 k Da,18.256 k Da,and 22.331 k Da,respectively,and each which has an RNA recognition motif RRM.In order to determine the interaction between Fbp1 and Grp1,first of all,we verified by yeast two-hybrid system and found that Fbp1 and Grp1 indeed interact.Then,we constructed the FBP1-FLAG::GRP1-HA strain for co-immunoprecipitation（Co-IP）,and we further proved that Fbp1 does interact with Grp1 through the Co-IP experiment.Fbp1 contains two domains,namely the F-box and the leucine-rich repeats（LRR）domains.In order to explore the interaction site of Fbp1 and Grp1,we constructed a segmented expression strain containing only the F-box domain of Fbp1.The F-box domain knockout strain was constructed earlier by our research group.In the background of these strains,Grp1-HA was expressed.Through the Co-IP experiment,the results showed that Fbp1 interacted with Grp1 through the LRR domain.To verify whether Grp1 is a substrate of Fbp1 and whether the stability of Grp1protein depends on Fbp1,we expressed Grp1-HA fusion protein in wild-type H99strain and fbp1Δmutant and tested the stability and ubiquitination level of Grp1protein in the background of these strains.The result showed that Grp1 was gradually degraded with the extension of culture time in the wild-type background but remained relatively stable in the fbp1Δmutant,indicating that the stability of Grp1 protein depends on Fbp1.The ubiquitination test found that compared with the Grp1ubiquitination level in the wild-type background,the Grp1 ubiquitination level was significantly reduced in the fbp1Δmutant background,indicating that the ubiquitination of Grp1 is directly regulated by Fbp1.To further study the function of Grp1,we constructed double knockout mutants of FBP1 and GRP1 genes and GRP1 overexpression strains.Later,we explored the changes of the three classic virulence factors of fbp1Δgrp1Δdouble mutant and GRP1^OE overexpression strains.It was found that the thickness of the polysaccharide capsule of C.neoformans decreased when GRP1 was overexpressed.The fbp1Δgrp1Δdouble mutant showed growth defects at 37℃,but both the fbp1Δgrp1Δdouble mutant and GRP1^OE strains do not affect on the production of melanin.Stress phenotype analysis showed that both fbp1Δgrp1Δdouble mutant and GRP1^OEoverexpression strains had growth defects in the medium containing SDS but not sensitive to Congo red and Calcofluor white（CFW）,which was consistent with the phenotype of fbp1Δmutant,indicating that both Fbp1 and Grp1 are necessary for the growth and development of Cryptococcus.Previous studies have shown that Fbp1 may affect the sexual reproduction of C.neoformans by regulating Grp1.To clarify this problem,we further explored the sexual reproduction of the fbp1Δgrp1Δdouble mutant and GRP1^OE overexpression strains of C.neoformans.The results showed that under the same induction conditions,compared with the wild-type strains,the fbp1Δgrp1Δdouble mutant was consistent with fbp1Δmutant and grp1Δmutant,and they could not produce basidiospores after mating,while the GRP1^OE overexpression strains did not mate.These results indicated that the normal expression of FBP1 and GRP1 is essential for the sexual reproduction of C.neoformans.Previous studies showed that the pathogenicity of C.neoformans was lost after FBP1 gene knockout.Therefore,we further tested the role of fbp1Δgrp1Δdouble mutant and GRP1^OE overexpression strains in the pathogenicity of C.neoformans.Through the results of the murine inhalational infection model experiment,we found that the pathogenicity of the fbp1Δgrp1Δdouble mutant and GRP1^OE overexpression strains were decreased significantly.Compared with the fbp1Δmutant,the deletion of GRP1 partially compensated for the lost virulence of fbp1Δmutant,and the overexpression of GRP1 also led to a significant reduction of the pathogenicity of C.neoformans.Observation of the histopathological section revealed that the lungs,brains,and spleens of the dead mice infected with fbp1Δgrp1Δdouble mutant,GRP1^OE overexpression strains,and H99 wild-type strains were damaged to varying degrees and contained a large number of cryptococcal cells.However,no cryptococcal cells were observed in the three tissues of the mice in the survival group,and the tissue structures were complete,indicating that both fbp1Δgrp1Δdouble mutant and GRP1^OE overexpression strains would be cleared by the immune system of mice at the late stage of infection.Then we collected tissues from mice inoculated with the strains mentioned above at different time points and found that cryptococcal cells were only counted in the lungs of infected mice on the first and third day.It shows that both fbp1Δgrp1Δdouble mutant and GRP1^OE overexpression strains would weaken the proliferation ability of Cryptococcus in the host and limited the early infection to the lungs.This may be the reason for the reduced virulence of fbp1Δgrp1Δdouble mutant and GRP1^OE overexpression strains.In conclusion,Cryptococcus Grp1 is a downstream substrate of Fbp1,which interacts with Fbp1 through the LRR domain of Fbp1,and the ubiquitination level of Grp1 is regulated by Fbp1.Through functional analysis of fbp1Δgrp1Δdouble mutant and GRP1^OE overexpression strains,we found that Fbp1 may regulate Grp1 through the ubiquitin-proteasome system,thereby affecting the sexual reproduction and virulence of C.neoformans.