Zinc Finger Protein Zfp2 Regulates Cell-cell Fusion And Pathogenicity In Cryptococcus Neoformans

Cryptococcus neoformans is a yeast-like human pathogenic fungus that exists widely in the natural environment.It can infect the human body through the respiratory pathway,mainly infecting people with immune deficiency such as AIDS patients or organ transplant recipients,and can also infect people with normal immunity.After C.neoformans enters the human body through the respiratory pathway,it will colonize the lungs,causing the host to suffer from cryptococcal pneumonia,and then spread to the brain through the blood circulation,resulting in a highly lethal cryptococcal meningitis.Currently,broad-spectrum antifungal drugs are the only means of clinical treatment of cryptococcosis.However,the lack of antifungal drugs and the continuous emergence of drug-resistant strains are two major challenges in the clinical treatment of cryptococcosis.C.neoformans has many virulence factors,including heat resistance,polysaccharide capsule,and melanin,which help C.neoformans to resist various adverse living conditions in the host.C.neoformans can produce smaller,faster-transmitting propagules through sexual reproduction and has also evolved to produce more pathogenic and drug-resistant strains.The study of the morphological development and pathogenic process of C.neoformans will enrich people’s understanding of the pathogenic mechanism of Cryptococcus and provide more potential therapeutic targets for the development of new antifungal drugs.It is also helpful to propose new and targeted strategies to overcome fungal infections,which have great scientific significance and potential clinical application value.Zinc finger proteins contain zinc finger domains and play an important role in eukaryotes.Different types of zinc finger proteins play different roles in fungal morphogenesis and pathogenesis.The Zfp2 protein in this study belongs to the C3HC4type zinc finger protein.We found that Zfp2 plays an important role in the formation of virulence factors,cell-cell fusion,and pathogenicity of C.neoformans.The results obtained are as follows:1.Identification of ZFP2 gene and construction of related strainsSequence analysis showed that the ZFP2 gene was 1517 bp in length and contained three introns,which could encode a C3HC4 type zinc finger protein Zfp2with a full length of 135 amino acids.We intend to explore the biological function of Zfp2 in C.neoformans by gene knockout,complementation,and overexpression.Therefore,we constructed zfp2Δmutant,zfp2Δ::ZFP2 complementation strain,and ZFP2^OE overexpression strain by biolistic transformation used for subsequent experiments.2.Phenotype analysis of ZFP2 gene knockout mutantsThe wild-type strain H99,zfp2Δmutant,zfp2Δ::ZFP2 complementation strain,and ZFP2^OE overexpression strain were tested for various virulence factors.The results showed that ZFP2 gene knockout not only affected the normal growth of C.neoformans but also affected its heat resistance and polysaccharide capsule formation.The results of the stress tolerance assay showed that ZFP2 gene knockout affected the integrity of C.neoformans cell wall and cell membrane.3.The effect of Zfp2 on the cell-cell fusion of C.neoformansThe zfp2Δmutant could not produce mating hyphae in mating experiments,and the ZFP2^OE overexpression strain could produce mating hyphae only in unilateral mating with KN99a.Through cell-cell fusion experiments,we found that ZFP2 gene knockout can block the cell fusion process of C.neoformans sexual reproduction.By detecting the expression changes of key genes in the pheromone response pathway during the cell-cell fusion process of C.neoformans,we speculate that Zfp2 may affect the process of cell-cell fusion by regulating pheromone response pathways.4.The effect of Zfp2 on the pathogenicity of C.neoformansWe tested the pathogenicity of H99,zfp2Δ,zfp2Δ::ZFP2,and ZFP2^OE strains using a mice inhalation infection model,and mice survival curves showed that both ZFP2 gene knockout and overexpression resulted in attenuated pathogenicity of C.neoformans.By taking samples from infected mice,both CFU statistics and histopathological sections showed that the proliferation ability of zfp2Δmutants and ZFP2^OE strains in mice lungs was lower than the wild-type strains.To further explore the reasons for the weakened proliferation of zfp2Δand ZFP2^OE strains in the lungs of mice,we carried out a Cryptococcus-macrophage interaction assay using J774 macrophage cells to test the survival of the Zfp2 related strains in macrophages.There was no significant difference between the zfp2Δmutant and the wild-type strain after being phagocytosed by macrophages;however,the ZFP2^OE overexpression strain showed significant growth defects after being phagocytosed by macrophages.In conclusion,the deletion of ZFP2 gene can cause the sexual reproduction of C.neoformans to be blocked in the cell fusion stage,and the pathogenicity of the strain is also significantly reduced.This suggests that the Zfp2 protein plays an important role in cell fusion and pathogenicity in C.neoformans.