Identification And Functional Study Of The Growth And Hyphae Formation Related Gene REI1 In Cryptococcus Neoformans

Background:Cryptococcus neoformans is a very important clinical opportunistic pathogen,which has the characteristics of aggregation and extensive distribution in the world.People with immunocompromise,such as patients with acquired immune deficiency syndrome(AIDS),cancers or organ transplant,at high risk of cryptococcosis.However,in recent years,the emergence of more and more people with non-immune deficiency infections has made cryptococcal infections more and more concerned.In recent years,C.neoformans’ morphogenesis and virulence related signaling pathways have attracted extensive attention.Cryptococcus neoformans is a yeast-hyphal dimorphic fungus,and its morphological transformation has profoundly affected and shaped the virulence of Cryptococcus.The yeast form is a highly virulent form of Cryptococcus,and it is also the main infection and pathogenic form of Cryptococcus,while Cryptococcus in a mycelial state will completely lose its toxicity.At the same time,the morphological transformation of Cryptococcus is closely related to sexual reproduction,and the genetic diversity of sexual spores produced by genetic recombination and meiosis during sexual reproduction is a very important factor in the emergence of highly virulent strains in clinical practice.The further study on the virulence and the morphology and development of C.neoformans and will help us to find new methods and approaches for the treatment of cryptococcosis.In this study,a new gene named REI1 was identified based on the previous research of the research group.This study provides a new idea for elucidating the sexual reproduction and pathogenic mechanism of C.neoformans.Methods:In this study,we would characterize the function of REI1 in the laboratory strains of C.neoformans XL280 alpha and a as background strains.We use Transient CRISPR-Cas9 Coupled with Electroporation(TRACE)System to construct rei1Δmutant strains,and then randomly insert a plasmid containing REI1’s own promoter and target gene sequence into the deletion strain to construct a complement strain.Wild-type XL280 and relative mutant strains were used to analyze the specific functions of REI1 in C.neoformans,which mainly included the following aspects:growth and development,environmental stress,virulence factors and sexual reproduction.Result:1.Identification and sequence analysis of REI1.A functionally unknown gene was isolated from the RNASeq result and named REI1.Sequence analysis revealed that the ecoding region of REI1 contains three introns and REI1 gene encodes 413-amino acids protein-Rei1.Domain analysis revealed that Rei1 protein contains two zinc finger domain.The phylogenetic tree showed that Rei1 protein was highly conserved.We analyzed the transcriptional expression of REI1 in the early mating stage and different developmental stages of C.neoformans by q RT-PCR.The results showed that transcriptional level of REI1 in the early mating stage was increased at first and then decreased,and which this trend is similar to that of yeast-to-hypha transformation related genes ZNF2,PUM1,etc.,suggesting the REI1 may play a role in the mating process of C.neoformans.2.Construction of REI1 gene knockout,and complementation strains.To further study the function of REI1,the alpha and a mating type rei1Δ mutant strain were generated by Transient CRISPR-Cas9 Coupled with Electroporation(TRACE)System.And then randomly insert a plasmid containing REI1’s own promoter and target gene sequence into the deletion strain by electroporation technology to construct a complement strain.3.Phenotypic analysis of rei1Δ mutant,and REI1 complement strains.1)By measuring the growth curve,we found that rei1Δ mutant strains needed longer time to enter the logarithmic growth phase and plateau phase than wild-type and complement strains.Temperature sensitivity experiment found that the deletion strain showed obvious growth defects under low temperature conditions,especially at4℃,the deletion strain hardly grew.2)Under the stress of Na Cl,Sorbitol,Congo red,SDS and other conditions of culture with different carbon sources,the growth of the deletion mutant showed no significant change compared with the wild-type strain.3)The classical virulence factor test found that the capsules induced by the REI1 deletion strain on DMEM medium were smaller than the wild type,and the growth was slightly weaker at 37℃.4)Mating assays showed that,whether unisexual reproduction or bisexual reproduction,the filamentation of the REI1 deletion strains are significantly inhibited,since they are almost no hypha growth,no basidia and no spore production.5)The results of real-time PCR showed that under the condition of hyphal induction,the peak expression of several important genes such as MAT2 and ZNF2 in the pheromone pathway after REI1 deletion was later than that of the wild type,and the expression of MAT2 increased,while the expression of ZNF2 and CFL1 decreased significantly.Conclusions:In this study,we identified a new gene REI1,which is related to the growth and filamentation of C.neoformans.By exploring the specific functions of the gene,we found that REI1 is involved in the regulation of the growth and sexual reproduction of C.neoformans,and may therefore change the virulence of the strain and weaken its pathogenicity.The function analysis of REI1 provides a new way to elucidate the sexual reproduction and pathogenic mechanism of C.neoformans.