Study On The Function Of A 14-3-3 Protein Bmh2 In Regulating Growth And Morphogenesis Of Cryptococcus Neoformans

Background:Cryptococcus neoformans is a common human fungal pathogen in clinic and is widely distributed in nature.It is easy to be inhaled by our human body through respiratory tract and colonized in lung.According to statistics,the death cases of Cryptococcus infection are up to more than 600,000 in the world every year,which making it one of the most pathogenic fungi with the highest morbidity and fatality rate.As a yeasty-hypha dimorphic fungus,the pathogenicity of C.neoformans is also closely related to its morphology.The yeast morphology is highly pathogenic,while the hypha form is avirulent.It was reported that a large amount of cell surface proteins and the secretory proteins will be synthesized and secreted during the yeast-hypha transition process.These proteins may be transported by vesicle to the cell surface or extracellular matrix to exert their corresponding functions.However,the secretion mechanism of these proteins and its relationship with morphologic transformation is not clear at present.Vesicles containing a lot of 14-3-3 protein,Bmh2,that associated with Cryptococcus virulence characteristics.This type of protein is usually combined with protein phosphorylation of serine or threonine,participating in a variety of signal transduction pathways,but the detailed functions of Bmh2 in C.neoformans are still unclear.Therefore,this study focused on the 14-3-3protein Bmh2 and conducted an in-depth study on its genetic function,which will help to provide new ideas for the elucidation of the morphological transformation mechanism and exploring for new anti-Cryptococcus strategies.Methods:1.Construction and verification of PCTR4-BMH2 mutants and complementary strains based on background strain-Cryptococcus neoformans serotype D strain XL280.Transient CRISPR-Cas9 combined with electroporation system（TRACE）was used to knock out and complement the target gene.2.Characterization of the cell morphology and sizeThose cell morphology were examined by the ordinary microscope and contrasted with those of WT,mutant strains and complementary strains.The cell diameter was measured by Image J and statistically analyzed by Graph Pad Prism8.3.Temperature and chemical sensitivity analysis（1）All strains were added in 20%glycerol and stored at-80°C,and activated on yeast extract-peptone-glucose（YPD）solid medium with agarose.Unless otherwise specified,all strains were routinely cultured at 30°C.（2）When detecting temperature sensitivity,we cultured the strains on YPD and YPD+25μMCu SO₄medium respectively,and monitored and compared the growth of the two groups of strains at different temperatures（25°C,30°C and 37°C）and photographed at 2-5 days.（3）When detecting chemical sensitivity,we cultured the strains on YPD+25μM Cu SO₄and YPD+25μMCu SO₄with various stress chemicals（KCl,Na Cl,Sorbitol,Congo Red,etc.）,and monitored and compared the two groups and photographed in1-3 days.4.Growth analysis（1）All strains were activated on YPD+25μMCu SO₄solid medium and cultured routinely at 30℃.（2）In order to draw the growth curve,the strains were cultured on YPD+25μMCu SO₄liquid medium and incubated in a constant temperature shaker at 30℃,and at a fixed time point,the bacteria solution was taken to determine OD_600.5.Unisex and hyphal formation testThe strains were cultured on V8+25μMCu SO₄solid medium.Unisexual reproduction was carried out under 25°C dark condition.Observe the production of hyphae under a microscope.Result:Compared with WT and complementary strains,the copper-induced conditional knock-out mutant strains are significantly restricted in growth,and their cell morphology is enlarged,and adhesions between cells are not easily separated.At the same time,the growth of hyphae is also significantly restricted,and the temperature and chemical sensitivity is higher.Conclusions:Our results suggest that BMH2 gene is closely involved into the cell morphogenesis and cell size,temperature sensitivity and response to environmental stress,and finally participates in the growth of Cryptococcus neoformans.More importantly,BMH2 gene is also closely related to the sexual reproduction and hyphal growth following sexual mating.This study clarified the function of BMH2 gene in the growth,sexual reproduction and morphological differentiation of Cryptococcus neoformans,and provided helpful ideas for exploring the pathogenesis,diagnosis and treatment of Cryptococcal pneumonia.