Pathogenicity And Metabolic Mechanism Of Cryptococcus Neoformans Deubiquitinating Enzyme And Combined Antifungal Of New Sensitizers

Objects Cryptococcus neoformans is one of the most common invasive pathogenic fungi in the clinic.Nowadays,the infection rate of cryptococcal bacteria is increasing year by year.Even if the application guidelines recommend drug treatment,the mortality rate will remain at around 20%.In the first part of the study,the ubiquitination enzyme Doa4 was used to regulate the virulence of cryptococcus from the perspective of pathogen-host interaction.In the second part of the study,according to the results of previous experiments,the infection status of cryptococcus was simulated,and metabolomics was used to explore the difference of non-targeted metabolites affected by the deletion of ubiquitination enzyme Doa4,and to explore the deubiquitinating enzyme Doa4.Mechanism pathways that affect virulence.In the third part of the study,it is proposed to determine whether there is synergistic effect between rattlerin and fluconazole against cryptococcal bacteria through the in vitro checkerboard method combined with drug sensitivity test.Method The ubiquitination enzyme Doa4 gene mutant was constructed by the principle of homologous recombination and gene knockout technology,combined with the in vitro phenotypic,cell and animal infection models to investigate the virulence of the deubiquitinating enzyme Doa4 against Cryptococcus.Regulatory utility.In the second part,the cryptococcal culture was compared with the conventional culture medium in vitro,and metabonomics detection technology was used to explore the difference of non-targeted metabolites of doa4? mutants from qualitative and semi-quantitative points.The cells were stained with cell wall to observe the effect of deubiquitinating enzyme Doa4 on the cell wall and the structure of polysaccharides on the capsule.In the third part,according to the American Clinical Laboratory Standardization Association M27-A3 guideline,it is proposed to determine whether there is synergistic effect between rattlerin and fluconazole against cryptococcal bacteria through the in vitro checkerboard method combined with drug sensitivity test.Results In the first part of the study,the ubiquitination enzyme Doa4 gene mutant strain and the restorer strain were successfully constructed.It was found that the deubiquitinating enzyme Doa4 is involved in various stress responses and virulence regulation of cryptococcus,and macrophages can enhance the mutant strain.Identification,phagocytosis and intracellular killing,and can regulate the adhesion and traversal of cryptococcus to the blood-brain barrier model;in animal infection experiments,the mutant strain significantly improved the survival rate of mice and reduced the bacterial load of various organs.In the second part,when the doa4? mutant strain was simulated tissue culture,there was a significant difference in intracellular sugar biochemical metabolism and synthesis and assembly metabolism of polysaccharides in cell wall and capsule compared with in vitro eutrophication.In the third part,the MIC value of rattlerin to 37 strains of cryptococcus is 32-256?g/ml.When combined with fluconazole,the FICI is ?0.50,which reduces the MIC value of fluconazole by 2-16 times.Conclusion 1.The deubiquitinating enzyme Doa4 gene may play an important role in regulating the non-classical virulence mechanism of cryptococcus neoformans.Second,the deubiquitinating enzyme Doa4 gene may play an important regulatory role in the process of immune adaptation and escape of cryptococcus neoformans.Third,the deubiquitinating enzyme Doa4 gene may regulate the virulence of cryptococcus neoformans by changing the intracellular sugar biochemical metabolism and energy metabolism,as well as the synthesis and assembly of the cell wall and capsule polysaccharides.4.Rattlerin has a certain antifungal effect on cryptococcus,and it can synergize with fluconazole to improve the sensitivity of fluconazole to cryptococcus.