| Background: With the spread of organ transplant surgery,immunosuppressive therapy and HIV,cryptococcal meningitis is becoming more common in immunocompromised patients,claiming hundreds of thousands of lives each year and becoming a major public health threat.In order to adapt to the environment and occupy different ecological niches,Cryptococcus neoformans can absorb and assimilate sulfides widely present in the environment,and synthesize methionine(Met)and cysteine(Cys)de novo in cells,further synthesizing polyamines,vitamins,coenzymes,glutathione,iron-sulfur clusters,etc.,to maintain normal physiological activities of cells.The sulfur metabolism process can be divided into 5 parts: sulfur uptake pathway,O-acetylserine pathway,O-acetylphoserine pathway,transulfurylation pathway and methyl cycle,most of them are quite different from human sulfur metabolism.The key metabolic enzymes and core transcription factors that regulate this process not only affect sulfur absorption and utilization,but also play a crucial role in the virulence and drug resistance of C.neoformans.Objective: Yeast one hybrid library containing only C.neoformans transcription factors(TF-onlyY1H)was constructed,and transcription factors that may be involved in regulating the sulfur metabolism of C.neoformans were obtained by library screening.Furthermore,the core transcription factor Cys3 of sulfur metabolism was determined by measuring the difference in sulfur source utilization of transcription factor deletion strains.By constructing mutant and complement strains,the effect of Cys3 on the virulence and drug resistance of C.neoformans was preliminarily studied,which provided a new idea for the subsequent development of antifungal drug targets.Method: 1.By comparing the existing transcription factor prediction database in C.neoformans,the candidate transcription factors to be amplified were determined.Design primers and amplify fragments.Then transcription factor fragments were cloned to p B42 AD plasmids by homologous recombination,and TF-only Y1 H library were constructed and stored.2.The promoter encoding the sulfate transporter SUL1 was cloned to pLacZi by homologous recombination and transformed into yeast cells EGY48 to form a bait strain.The autoactivation effect of bait strain was detected by blue-white spot screening.3.Transcription factors directly bound to SUL1 promoters were screened by TF-only Y1 H library,the growth of candidate transcription factors deletion strains under Cys and sulfate conditions was measured,and the core transcription factors of C.neoformans sulfur metabolism were determined.4.RT-qPCR was used to determine the expression pattern of Cys3.5.The function of Cys3 was verified by constructing mutant and complement strains,and its growth curves under different sulfur source conditions were tested to determine the effect of Cys3 on cell utilization of sulfur source.Combined with transcriptome sequencing analysis,the molecular mechanism of Cys3 regulation of sulfur metabolism was proposed.6.By detecting the growth of cys3Δ and cys3Δ+CYS3 under various stresses,the effect of Cys3 on cells coping with environmental stress was explored.7.The effect of Cys3 on the virulence of C.neoformans was determined by detecting virulence factors and establishing a mouse nasal infection model.Result: 1.By comparing Transcription factors databases and reviewing literature,301 candidate Transcription factors were identified.After 5 rounds of independent cloning,271 plasmids were constructed successfully and sequenced correctly,accounting for 90.03% of the total.The most complete TF-only Y1 H library in C.neoformans was successfully established.The library includes 32 predicted transcription factor families,accounting for 94.12% of the total.23 of these families(67.65% of the total)have 100% clonal coverage,and only 2 families(Gnt R-like bacterial transcription factor,CCR4-NOT transcription complex)do not appear in the collection.2.Autoactivation assays of the bait strain revealed no self-activating activity of the SUL1 promoter within yeast cells.3.After the initial screening and rescreening of the Y1 H library,33 transcription factors that can bind to the SUL1 promoter were obtained,including 9 “Very Strong”,5“Strong”,19 “Weak”,and 0 “Medium”.The growth curve of candidate transcription factors deletion showed that the ability of CNAG＿06223Δ to utilize Cys and sulfate was significantly enhanced(P <0.001),comparing with WT type.The utilization capacity of CNAG＿04798Δ was significantly decreased(P <0.001).The ability of CNAG＿03366Δ and CNAG＿00841Δ to utilize sulfate decreased(P <0.05),while there was no difference in growth under Cys conditions.The growth capacity of CNAG＿04090Δ in sulfate is significantly reduced(P <0.001),and the growth rate under Cys is slowed down.CNAG＿05222Δ growth slowed down in both sulfur source environments and entered the plateau later.Among them,CNAG＿04798-encoded Cys3 had the greatest effect on the uptake and utilization of organosulfur sources by cells.4.RT-qPCR assays showed that CYS3 transcriptional expression was regulated by sulfur concentration,independent of the type of sulfur source contained in the environment.5.The growth curves of cys3Δ under different sulfur sources showed that cys3Δcould only be grown in medium with 10 m M Cys added,and could not utilize other tested sulfur sources.Transcriptome sequencing analysis revealed that this growth difference stems from Cys3’s extensive regulation of key enzymes in the process of inorganic sulfur assimilation of C.neoformans.6.Observing the growth of cys3Δ under various stress stresses,it was found that compared with WT type,cys3Δ was significantly sensitive to oxidative stress(P <0.01),sensitive to heavy metal stress(P <0.05),and insensitive to salt ion stress.At the same time,cys3Δ has a significantly increased sensitivity to fluconazole,while it is insensitive to amphotericin B.7.Virulence factor test results showed that Cys3 could significantly affect the capsule thickness of cells,but did not affect the formation of melanin and normal growth at 37 °C.In mouse nasal infection models,Cys3 was found to significantly affect the colonization ability of C.neoformans in the brain and lungs of mice,and knocking out CYS3 significantly prolonged the survival time of infected mice.However,when cys3Δ is in sulfur-restricted environment for a long time,there is a chance to restore some growth capacity and pathogenicity of WT type.Conclusion: The TF-only Y1 H library of C.neoformans is an effective tool for mining novel transcription factors.Cys3 screened in this study extensively regulates the expression of key enzymes and transporters in the sulfur metabolism process of C.neoformans,which significantly affects the sensitivity of C.neoformans to oxidative stress,heavy metal stress and fluconazole,and also affects the capsule thickness of cells and their colonization ability in the brain and lungs of mice.The survival time of mice infected with cys3Δ strains is significantly prolonged.These results suggest that Cys3 has the potential to be used as a target for novel antifungal drugs. |
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