Identification Of Sulfur Transporters And Their Role In Pathogenicity In Cryptococcus Neoformans

Background:Cryptococcus neoformans is an important opportunistic fungal pathogen that causes cryptococcal meningitis,a serious threat to human health,causing more than 230,000 deaths each year.Sulfur is an essential nutrient for the survival and reproduction of microorganisms.The host body is rich in various sulfur sources,such as sulfate and sulfur-containing amino acids.Pathogenic fungi adopt a variety of sulfur-source acquisition strategies to ensure the supply of this essential nutrient.Sulfur transporters play a key role in the process of sulfur uptake,and their expression is strictly regulated by cellular sulfur levels.Given the important physiological functions of sulfur compounds,the analysis of the function and regulatory mechanism of sulfur transporters may provide new ideas and drug targets for the prevention and control of cryptococcosis.Objective:This study aims at identify of sulfur transporters of inorganic and organic sulfur in C.neoformans.To explore the impact of sulfur transporters on the virulence of C.neoformans.Understanding the regulation mechanism of transcription factor Cys3 on sulfur transporters is instrumental in enriching the molecular mechanism of fungal sulfur regulation metabolism.Method:1.The physiological state of cells treated with long-time sulfur starvation was observed and the transcriptome of short-time sulfur starvation was sequenced.The importance of sulfur to C.neoformans was assessed by testing the effects of sulfur starvation on the physiology of the cell.2.The candidate sulfur transporters were identified by RNA-seq combined with biosynthesis analysis.The knockout and complement strains were constructed to observe the growth phenotype of the sulfur transporters on the sole sulfur source,and the function of the sulfur transporters was initially studied.It revealed the gene expression patterns of genes of sulfur transporters by using qRT-PCR and luciferase reporter gene system.3.The fusion expression strain of the sulfur transporter and mCherry was constructed,and the subcellular localization of the sulfur transporter was observed by fluorescence microscopy.4.The virulence factors and stress phenotypes of mutant strains were tested to explore the effect of sulfur transporters on the expression of virulence factors and their tolerance to various stresses.To investigate the effect of sulfur transporters on the pathogenicity of C.neoformans by using the Galleria mellonella infection model and mouse infection model.5.RNA-seq was used to compare the expression patterns of wild-type and cys3Δ in a condition of sulfur starvation and enough sulfur.To identify genes that may be regulated by CYS3 by differentially expressed genes analysis.The conserved motifs analysis was used to analyze possible Cys3 binding sites.To explore the molecular mechanism of Cys3 regulating the expression of sulfur transport genes by yeast single hybridization.Result:1.It stops cell growth and reduce survival and cell diameter when suffering long-term sulfur starvation.It revealed the gene expression pattern in response to sulfur starvation by RNA-seq that is general transcriptional upregulation of sulfur acquisition and sulfur metabolism while the transcriptional downregulation of genes related to amino acid synthesis,translation and basal metabolism.2.The phenotype of deletion mutants and complementary strains with inorganic sulfur source as the sole sulfur source revealed that sul1Δ,sul1Δ soa1Δ and sul1Δ soa1Δ sua1Δ had obvious growth defects when sulfate was the sulfur source,and sul1Δ and sul1Δ soa1Δ had obvious growth defects when sulfite or sulfide or taurine was the sulfur source.Phylogenetic analysis Sua1 is distantly related to known sulfate transporters in fungi and is widely present in Ascomycetes and Basidiomycetes.qRT-PCR and luciferase reporter gene system showed that the expression of SUL1,SOA1 and SUA1 was induced by sulfur starvation,induced by sulfate and correlated with sulfate concentration.Fluorescence microscopy showed that Sul1 and Soa1 were mainly located in the plasma membrane.3.The phenotype of deletion mutants and complementary strains with organic sulfur source as the sole sulfur source revealed that mup1Δ had obvious growth defects when cysteine or homocysteine or cystine or cystathionine was the sulfur source,and the growth of sul1Δ was significantly slower than wild type when methionine was the sulfur source.Moreover,the growth of complementary strains could recover.qRT-PCR and luciferase reporter gene system showed that the expression of MUP1 was induced by sulfur starvation,induced by cysteine and correlated with cysteine concentration.Fluorescence microscopy showed that Mup1 was mainly located in the plasma membrane.4.The stress phenotype showed that sul1Δ growth was reduced under 50 μM heavy metals cadmium and chromium.The GSH content of sul1Δ under heavy metals was lower than the wild type.In the mouse infection model,the survival time of sul1Δ soa1Δ sua1Δ and sul1Δ was prolonged,and there was no significant difference between the two groups.The growth phenotype in vitro and competition experiments in vitro showed that the ability of growth and survival in sul1Δ was weaker than the wild type.5.RNA-seq analysis showed that SUL1,SOA1,SUA1 and MUP1 were significantly up-regulated when wild-type was in sulfur starvation but not differentially expressed when cys3Δ was in sulfur starvation.The conserved motifs analysis showed that the promoter regions of SUL1,SOA1 and SUA1 contained the possible Cys3 binding site AGATGGCGTGATBGA,but MUP1 did not.Yeast single hybridization showed that SUL1,SOA1 and SUA1 promoters could bind to Cys3,but the MUP1 promoter could not.Conclusion:Sulfur is an essential nutrient for C.neoformans.Sulfur starvation affects the physiological state of the cell.Sul1,Soa1 and Sua1 are all sulfate transporters in C.neoformans.Sul1 is the major sulfate transporter,Soa1 and Sua1 are the minor sulfate transporters.The transport substrates of Sul1 of the Sul P family and Soa1 of the MFS superfamily include sulfate,sulfite,sulfide and taurine.Sua1 of the MFS superfamily may be a sulfate-specific transporter and may represent a new class of sulfate transporters.Mup1 is a transporter of cysteine,homocysteine,cysteine,and cystathionine,whereas the sulfate transporter Sul1 affects methionine utilization.Sul1,Soa1 and Mup1 are mainly located in the plasma membrane,and play a transport function from extracellular to intracellular.The deletion of SUL1 reduces the content of GSH,which leads to decrease tolerance to cadmium and chromium.The pathogenicity of sul1Δsoa1Δ sua1Δ which is unable to utilize sulfate is reduced in the mouse infection model,mainly due to the reduced growth and survival ability due to the deletion of SUL1.The DNA binding site of transcription factor Cys3 may be AGATGGCGTGATBGA.Three sulfate transporters containing this sequence bind to Cys3 and are directly regulated by Cys3.However,the cysteine transporter Mup1 without this sequence does not bind to Cys3 and is indirectly regulated by Cys3.