Regulation Of N-acetylglucosamine Catabolism Pathway In Cryptococcus Neoformans And Its Influence On Pathogenicity

Background:Cryptococcosis has become a common invasive fungal disease threatening human health with the epidemic of AIDS since the 1980s.Cryptococcal meningitis causes approximately 15%of AIDS-related deaths worldwide.Cryptococcus neoformans is the predominant cause of HIV-associated adult meningoencephalitis in sub-Saharan Africa.As an environmental fungal pathogen,Cryptococcus exists in different natural niches around the world and has strong adaptability to various environments.Cryptococcus neoformans can uptake and catabolize a variety of carbon sources as energy sources for life activities.Amino sugars are important sources of nutrients in glucose-limited environments.GlcNAc,the monomer of the second-largest carbohydrate chitin,is a component of many life structures and has various physiological functions.GlcNAc is also involved in regulating different fungal biological processes,including morphological transformation,virulence,oxidative stress,and antifungal sensitivity.Fungi can respond to carbon sources in the environment by triggering different signal transduction pathways,allowing them to adapt to environmental pressures and different ecological niches.Objective:In this study,we identified the GlcNAc catabolism gene cluster in Cryptococcus neoformans.We discovered a novel transcription factor Gcr1 that regulates this pathway,and we preliminarily clarified the molecular mechanism of its regulation.Improving the understanding of the GlcNAc catabolism pathway in Cryptococcus neoformans will facilitate the development of new anti-cryptococcal infection strategies.Method:1.RNA-seq was used to compare the differentially expressed genes in Cryptococcus neoformans with or without GlcNAc.Genes related to GlcNAc transport and catabolism were found through sequence alignment analysis.2.The gene expression patterns of putative GlcNAc transporter and catabolism genes were analyzed by RT-qPCR,and their functions were verified by constructing deletion strains and phenotypic experiments.3.The conserved active sites of GlcNAc kinase in Hxk3 were determined by amino acid sequence analysis.Hxk3 was expressed in Escherichia coli heterologously.The kinase activity of Hxk3 was determined by HPLC and coupled enzyme ATPase assay,and its enzymatic kinetic parameters were determined.4.The transcription factor Gcr1 that regulates GlcNAc catabolism was found through phenotypic screening,and its function was verified by constructing deletion strains and complemented strains,and the expression pattern of GCR1 was determined by RT-qPCR.Phenotypic experiments and RNA-seq were used to demonstrate the specificity of Gcr1 regulation.5.The functional domains of Gcr1 were analyzed using bioinformatics tools.Gcr1direct binding promoters and the recognition motif of Gcr1 were identified by yeast one-hybrid assay and ChIP-PCR/ChIP-seq assay.The dimerization domain of Gcr1 was determined by yeast two-hybrid assay.The subcellular localization of Gcr1 was determined by fluorescence localization.Phenotypic experiments were performed to identify small molecules that might directly bind to Gcr1.6.The conservatism of the GlcNAC catabatic gene cluster in Tremellales was verified by phenotypic experiments after knocking out and complementing the homologous genes of GCR1 and HXK3 in Cryptococcus gattii.7.The effect of Gcr1 on the pathogenicity of Cryptococcus neoformans was determined by the nasal inhalation infection mouse model.Result:1.RNA-seq identified 12 GlcNA-induced genes and 10 GlcNAc-repressed genes.These GlcNAc-induced genes included GlcN-6-P deaminase（Nag1）,GlcNAc-6-P deacetylase（Dac1）,GlcNAc transmembrane transporter（Ngt1）,and a novel GlcNAc kinase with low identity to the GlcNAc kinases of Candida albicans and Trichoderma reesei.2.The results of RT-qPCR showed that the expression levels of NAG1,NGT1,DAC1,and HXK3 significantly increased after GlcNAc induction.And the gene expression pattern of HXK3 was consistent with NAG1,NGT1,and DAC1.Deletion mutants of each of these four genes showed distinct growth defects on the medium supplemented with GlcNAc as the sole carbon source,while the growth of the deletion mutants showed no difference from that of the wild type on the medium supplemented with glucose as the sole carbon source.3.Multiple sequence alignment revealed that Hxk3 also had the same conserved GlcNAc binding residues as other GlcNAc kinases.In vitro enzymatic reactions and coupled enzyme ATPase assay also indicated that Hxk3 had high substrate specificity for GlcNAc.4.The gcr1Δdeletion strains had growth defects on the medium supplemented with GlcNAc as the sole carbon source,but they grew well on the medium supplemented with other carbon sources.The GCR1 complemented strains restored the ability to grow on the medium with GlcNAc as the sole carbon source.The RT-qPCR assay demonstrated that GCR1 expression was regulated by glucose repression.RNA-seq showed that the expression patterns of the gcr1Δdeletion strain were highly consistent under the two carbon source conditions,proving that Zn₂C₆ zinc cluster protein Gcr1 is a transcription factor specifically regulating the catabolism of GlcNAc.5.Yeast one-hybrid and ChIP-PCR/ChIP-seq experiments showed that Gcr1 plays a regulatory role by recognizing a novel DNA recognition motif（CGGGGAAANCGGCG）on the promoter of GlcNAC transport and catabolism genes.Yeast two-hybrid experiments showed that Gcr1 played a regulatory role in the form of a homodimer.Cellular sublocalization analysis showed that Gcr1 was specifically localized in the nucleus,and the localization of Gcr1 was not affected by carbon sources.In addition,Gcr1 may also need to bind GlcNAc catabolic intermediates for full function.6.There was no significant difference in the survival time of mice intranasally infected with gcr1Δdeletion and GCR1 complemented strains.Conclusion:The GlcNAc catabolic gene clusters exist in Cryptococcus neoformans,which are positively regulated by transcription factor Gcr1.Gcr1 binds to the promoter containing the Gcr1 recognition motif through the zinc cluster domain and binds to the small ligand molecule in the form of homodimer to play the transcriptional activation function to activate the expression of target genes.Gcr1 and GlcNAc catabolic gene clusters are conserved in Tremellales and they did not affect the pathogenicity of Cryptococcus neoformans in mice.