Effect Of Zinc Cluster Transcription Factor Gcr1 On Pulmonary Colonization Of Cryptococcus Neoformans And Its Molecular Mechanism

Background:Cryptococcus is one of the three major human invasive fungal pathogens,with Cryptococcus neoformans（C.neoformans）being the primary causative agent.After inhalation of spores or dried yeast cells,Cryptococcus neoformans is the first to establish an infection in the lungs.In immunocompetent people,it is often seen as a latent infection in the lungs with no significant clinical symptoms,whereas in immunodeficient or immunocompromised patients,Cryptococcus neoformans can colonies the lungs and then proliferate and spread throughout the body,leading to fatal cryptococcal meningitis.How does Cryptococcus neoformans sense the source of nutrition and thus regulate its own life activities?N-Acetylglucosamine（GlcNAc）,nature’s second most abundant glycoglycan monomer,is involved in the regulation of several biological processes in fungi,including morphological transformation,virulence,oxidative stress and response to antifungal agents,and is involved in the regulation of cell death in several fungi.Cryptococcus neoformans can use GlcNAc as a carbon or nitrogen source for its growth.In the human lung,mucus,a component of the first line of defense,is rich in GlcNAc and,in addition,commensal microorganisms in the lung can be an important source of GlcNAc.Objective:The transcription factors regulating GlcNAc transport and metabolism genes in Cryptococcus neoformans were identified.The effect of transcription factor GlcNAc catabolism regulator 1（Gcr1）on the physiological function of Cryptococcus neoformans was investigated through an animal experiment in a mouse nasal aspiration model,and the molecular mechanism of Gcr1 regulation was elucidated.This thesis provides a preliminary investigation into the mechanism of Cryptococcus neoformans lung colonization,and lays a solid foundation for the identification and diagnosis of Cryptococcus lung.Method:1.Identification of transcription factors that may regulate GlcNAc transport and catabolic genes by genomic location analysis of the GlcNAc gene cluster.Growth phenotypes of deletion strains in GlcNAc were constructed and tested and the expression levels of GlcNAc catabolic genes were analyzed by Real Time Quantitative PCR（RT-qPCR）to identify transcription factors regulating GlcNAc transport and catabolic genes.2.Analysis of the individual functional domains of Gcr1 using various tool sites and typical zinc cluster transcription factor structures.3.The effect of Gcr1 on the colonization of Cryptococcus neoformans in the lung was investigated in a mouse nasal aspiration infection model,and its colonization in the lung was investigated using knockout-free strains with deletions of the functional structural domains（DNA-binding domain（DBD）and ligand-binding domain（LBD））of Gcr1,and the reasons for the effect of Gcr1 on the colonization of Cryptococcus neoformans in the lung were investigated based on stress phenotype experiments and growth phenotype experiments.4.Gcr1 was proposed as a ligand-dependent transcription factor based on gene overexpression,RT-qPCR and the small molecule ligand of Gcr1 was identified by SPR.5.The key amino acids bound to the ligand in the structural domain of the Gcr1ligand were predicted by molecular docking,the conserved nature of the key amino acids was analyzed based on sequence alignment,and the growth phenotype,reporter gene expression and protein expression were determined after the alanine mutation was constructed by seamless cloning to verify its effect on Gcr1 function.6.The proteins that may interact with Gcr1 were identified using Immunoprecipitation-Mass Spectrometry（IP-MS）after incubation under two different conditions and confirmed by yeast two-hybrid,reporter gene expression studies.Result:1.Combined with the results of previous studies and the analysis of GlcNAc gene cluster genome distribution,it was determined that Gcr1 is a transcription factor regulating GlcNAc transport and metabolism gene cluster.Without GCR1,Cryptococcus neoformans could hardly grow in GlcNAc,and GlcNAc gene cluster gene expression was significantly down-regulated,further confirming that Gcr1 is the core transcription factor regulating GlcNAc transport and metabolism gene cluster.2.Gcr1 contains 1068 amino acid residues,of which the DNA-binding structural domain（DBD）is Cys₂₃₆-Cys₂₆₅ with a conserved Cys X₂Cys X₆Cys X₈Cys X₂Cys X₆Cys motif;the Nuclear Localization Signal（NLS）sequence is located at Gly₃₅₀-Ser₃₆₁;Gln₂₇₇-Asn₃₀₉ is probably the coiled helix domain,which acts as a hydrophobic structure can form a homodimer with another Gcr1 protein;Leu₅₉₀-Ser₉₁₈ is the ligand structural domain（LBD）of Gcr1;the activating structural domain of Gcr1 is Asp₁₀₁₂-Gln₁₀₄₃.3.Lung colonization of gcr1Δ,gcr1Δ（DBD）,gcr1Δ（LBD）and dac1Δ（GlcN-6-P deacetylase）decreased on the 7th day after infection.The growth of gcr1Δwas consistent with wild type（WT）in YPD plates containing Congo Red and Sodium Dodecyl Sulfate（SDS）.Cryptococcus neoformans can use mucin and its glycosyl monomers（N-acetylglucosamine,Galactose,Mannose,Ribose,N-acetylgalactose,Fucose and Sialic acid）to grow,among which GlcNAc and Galactose can be effectively used by Cryptococcus neoformans.The growth of gcr1Δwas deficient in the medium with mucin as the sole carbon source.4.Even if Gcr1 is overexpressed,it can play a regulatory role only in the presence of GlcNAc,which causes the relative expression level of NAG1（GlcN-6-P deaminase）and DAC1 to be up-regulated.After deletion of DAC1,the relative expression level of NGT1（GlcNAc specific transporter）and NAG1 decreased,suggesting that GlcN-6-P may be required for Gcr1 to play a regulatory role.SPR experiments show that Gcr1 can bind to GlcN-6-P with an affinity constant of 386 m M.These results indicate that GlcN-6-P is a small molecular ligand of Gcr1.5.Five possible key amino acids(Ser₆₀₂,Tyr₆₀₅,Leu₇₆₄,Thr₇₇₁,His₈₂₈)were identified by docking the simulated three-dimensional structure of LBD with small molecules and LBD sequence alignment of Gcr1 homologous proteins of Tremellales.After constructing alanine mutations one by one,the growth phenotypes were determined and Tyr₆₀₅ and Leu₇₆₄ mutations showed growth defects and reduced reporter gene expression.WB analysis showed that a single mutation did not affect protein expression,while Gcr1 was not expressed after Tyr₆₀₅/Leu₇₆₄ double mutation.6.About 20 proteins could interact with Gcr1 under YPD,YNB+Gal+GlcNAc;AH109 of pGAD7（Gcr1）and p GBKT7（CNAG＿02578）plasmids were not grown in yeast two-hybrid test.Expression analysis of reporter genes revealed that Hxk3（GlcNAc kinase）affects the regulatory function of Gcr1.Conclusion:Gcr1 is a central transcription factor in Cryptococcus neoformans that regulates the transport and catabolism of GlcNAc.Gcr1 may reduce Cryptococcus neoformans colonisation of mouse lungs by affecting the catabolism of GlcNAc,or by not fully utilising lung mucin to support its growth.The regulatory effect is also influenced by Hxk3.