The Helicase Protein Dna2 Regulates Sexual Reproduction And Pathogenicity Of Cryptococcus Neoformans

Cryptococcus neoformans is an encapsulated yeast-like fungal pathogen that can infect AIDS patients,organ transplant recipients,and immunocompromised populations.C.neoformans enters the lungs through the respiratory tract in the form of dried yeast cells or spores and further penetrates the blood-brain barrier to infect the central nervous system,causing fatal cryptococcal meningitis,killing nearly 200,000 people worldwide every year.The polysaccharide capsule is a unique virulence factor of C.neoformans,which acts as a thick protective layer on the outside and helps Cryptococcus evade the attack from the host.Besides the capsule,C.neoformans has many other virulence factors,including the ability to grow normally at 37oC and the production of melanin,urease,and phospholipase.C.neoformans usually reproduces asexually by budding,but it can also reproduce sexually under particular circumstances.The sexual reproduction of C.neoformans includes bisexual reproduction ofα-a and unisexual reproduction ofα-α,both of which go through the yeast cells,hyphae,basidium,and basidiospore formation.Diverse reproductive methods enable Cryptococcus to better adapt to various changes in the environment and facilitate population variation and evolution.Due to the lack of effective and widely available antifungal drugs in the clinic and the problems such as drug resistance that have emerged continuously in recent years,the current clinical treatment of cryptococcosis is ineffective.Therefore,the development of new drugs has become an urgent problem for scientists to solve.Furthermore,the current understanding of the interaction between pathogens and hosts is too shallow to provide effective guidance for clinical treatment.Therefore,basic research on the morphological development and pathogenic mechanism of Cryptococcus is particularly important.Helicases are a class of proteins that catalyze the unwinding of double-stranded DNA or RNA during DNA or RNA replication.Helicases are ubiquitous in eukaryotes and Dna2 is a helicase protein that is ubiquitous and highly conserved in eukaryotes.Dna2 has the activities of helicase,nuclease,and ATPase.It has been well-studied in many eukaryotes such as Saccharomyces cerevisiae,Schizosaccharomyces pombe,Mus musculus,Gallus gallus,and Homo sapiens.Dna2 is involved in a series of the metabolic activities,including Okazaki fragment processing,maintenance of replication fork,mitochondrial structure stability,DNA damage repair,and so forth.However,the Dna2 has not yet been studied in C.neoformans.In this study,we identified a predicted helicase protein Dna2 in C.neoformans and carried out the functional analysis,aimed to elucidate the role of Dna2 in sexual reproduction and virulence of C.neoformans.We got the following results:1.Identification and analysis of the spatiotemporal expression pattern of DNA2The DNA2 gene is 5149-bp long,including 12 introns and 13 exons,encoding a Dna2 protein with 1419 amino acids.The protein domain analysis showed that Dna2contains the DNA2 domain,Cas＿Cas4 domain,Helicase＿Rec D domain,AAA＿11,and AAA＿12 domains,indicating the Dna2 protein has helicase,nuclease,and ATPase activities.Multiple sequence alignment showed the sequence similarity was 37.53%between the Dna2 protein of C.neoformans and homologous proteins in Candida albicans,S.cerevisiae,and S.pombe.To explore the spatiotemporal expression of the DNA2 gene,we constructed P_DNA2-m Cherry fluorescent strain and GFP-Dna2 fluorescent strain.We set up the bilateral mating of P_DNA2-m Cherry strains and observed the fluorescence signal using confocal microscopy.Red fluorescence signal could be detected in yeast cells,dikaryon hyphae,and basidia,but no red fluorescence signal was detected in spores,implying that the DNA2 gene may not be expressed in basidiospores.By observing the fluorescence signal of GFP-Dna2 and DAPI staining,we found that Dna2 was localized in the nucleus,and the localization could not be affected in various stress conditions.It is located in the nucleus in yeast cells,dikaryon hyphae,and basidia,but no green fluorescence signal was detected in spores,which reconfirmed the speculation that DNA2 could not be expressed in basidiospores.Then the mating of wild-type H99 and KN99a strains were set up and mating mixtures were collected at different time points.The expression level of DNA2 were analyzed by RT-q PCR and the results showed that the expression of the DNA2 gene showed a periodic change of―first decrease and then increased‖in the early mating stage of C.neoformans.We speculated that Dna2 is actively involved in the metabolic activity of C.neoformans in the early mating period.2.Construction and phenotypic analysis of DNA2 knockout,complementation,and overexpression strainsTo further study the function of Dna2,we constructed the DNA2 knockout mutants,complementation strains,and overexpression strains.We first tested the production of the classical virulence factors of the above-mentioned strains,including capsule formation,melanin production,and resistance of high temperature（37oC）.The results showed that the capsule thickness of the dna2Δmutant and the DNA2^OE overexpression strains were significantly reduced compared to the wild-type H99.However,there was no significant difference in the resistance of high temperature and melanin production between the above-mentioned strains.The results of the growth of each cryptococcal strains under stress conditions showed that the dna2Δmutant was sensitive to HU and MMS,and the DNA2^OE overexpression strain was sensitive to HU.Therefore,we speculated that Dna2 may be involved in DNA replication and alkylation damage repair.Next we set up the matings of each cryptococcal strain on MS media,and the results showed that bilateral mating of dna2?mutants failed to produce basidiospores compared with wild-type strains.To determine why the dna2?mutants failed to generate basidiospores,we fused the nuclear localization protein Nop1 with the red fluorescent protein m Cherry to monitor the fungal nuclei positioning at different stages of sexual reproduction in C.neoformans.Two nuclei and one single fused nucleus could be observed in the basidia of both the wild-type and dna2?mutant strains.However,no four nuclei be observed in the basidia of the dna2?mutant strains,implying that the process of meiosis after nuclear fusion cannot normally occur.Therefore,we concluded that the reason why dna2Δmutant strains do not produce spores during bilateral mating is because the nucleus of the basidium cannot undergo meiosis to produce four basidiospores after the fusion of the nucleus during the mating process.The results indicated that Dna2 is involved in the meiosis in C.neoformans.3.The role of Dna2 on the virulence and pathogenicity of C.neoformansIn a murine inhalation model of cryptococcosis,we tested the virulence of the wild-type H99,dna2Δmutant,DNA2 complemention,and overexpression strains.The results showed that mice infected by the dna2?mutant survived longer than that of the wild-type H99,indicating that the virulence of dna2Δmutant was significantly attenunated.To explore the reason for the attenunated virulence of the dna2Δmutant strain,we infected mice with the dna2Δmutant strain and dissected the mouse organs of the lung,brain,and spleen at different time points.The fungal burden showed that the proliferation ability of dna2Δmutant in mice was significantly lower than that of the wild type.The results of histopathological sections showed that no yeast cells and lesions appeared in the brain and spleen of the mice infected by the dna2Δmutant at three time points.Therefore,we speculated that the decreased pathogenicity of dna2Δmutant was due to its reduced proliferation and dissemination in the host.In conclusion,we identified a predicted helicase protein Dna2 in C.neoformans and found it plays an critical role in the sexual reproduction and virulence of C.neoformans.