The Analyses Of Associated Genes Influencing The Virulence Differences Between Two Genotypes Of Cryptococcus Neoformans Var. Grubii

Objectives To investigate the virulence-associated genes in Cryptococcus neoformans var. grubii between two groups of different genotype strains, MLMT-13 and MLMT-36, and discuss the possible function of these genes which may cause the virulence difference. Methods 1. The genotypes of 10 environment isolates and 10 clinical isolates were analyzed with the multilocus sequence typing （MLST） method and the matting types of these strains were identified.2. According to the existing genome database, specific primers were designed to amplify and sequence the fragments of virulence-associate genes. Each gene was compared between the two groups of microsatellite genotype strains to analysis single nucleotide polymorphisms （SNPs） by sequence alignment. The non-synonymous SNPs （nsSNPs） which can change amino acid were filtered out. The corresponding proteins were analyzed and their structure and function were predicted.3. The high virenlent strain in clinical group and the less virenlent strain in environment group were selected for performing the whole genome resequence analyses. The sequencing results were comprehensively analyzed by the method of comparative genomics. Genetic variations were extensively screened through strategies of nsSNPs, nonsense SNPs and the InDels which caused frameshift mutations.4. The filtered genes were sequenced in all 20 testing strains. Whole RNAs were extracted and then the full-length cDNAs were sequenced with rapid amplification of 51 and 3’cDNA ends （RACE） method.5. A target gene knockout cassette which contains the nourseothricin-resistant marker （NAT） gene and 1-kb flanking regions of CNAG₀1032 was generated. After then, the cassette was introduced biolistically into C. neoformans cells. Transformants were screened on YPD medium with nourseothricin, and the proper integration event was comfimed by PCR and sequence. Results 1. The multilocus microsatellite genotype of all the 10 environment isolats was MLMT-13 in previous experiment and the MLST type of there strains was ST-15 in this experiment. Similarly, all the MLMT-36 clinical isolates belong to ST-32. And all the strains were MATa.2. Eleven relevant genes were sequenced successfully. Compared sequence data between the two genotype strains, besides two genes were identical, the other nine genes were detected with 1-4 SNPs respectively. And two genes turned out to be influenced with nsSNPs. The products of these two genes were protein Hst302 which was the family member of silent information regular 2 （Sir2） and the UV excision repair protein Rad23. In addition, the phylogenetic analysis revealed that most of the Rad23 proteins found in basidiomycetes are generally more homologous to the orthologs in human than to those found in other fungal phyla.3. By whole genome resequencing, valid data with high coverage was obtained in both environmental strain IFM56731 and clinical strain IFM56800. The data of InDel and SNPs were analyzed respectively. Six genes were chosen for further analysis through strategies of nonsense SNPs and the InDels causing frameshift mutations.4. The six genes were amplified and sequenced in all studied strains. And three genes were sequenced with cDNA. Ultimately, the location and structure of gene CNAG₀1032 was determined and nonsense mutation locus was verified to present in the actual mRNA.5.20 of 24 positive transformans were proved to knockout CNAG₀1032 gene successfully and the 1st transformant was finalized as the gene CNAG₀1032 deletion strain. Integrated selection with three PCR amplifications and four groups of medium containing antibiotics, three transformations which integrate expression vector was successfully achieved and could be steadily hereditary. Conclusions 1. The MLST results are highly consistent with MLMT results in previous experiment which reveals that both molecular typing methods are high resoluted and stable on classification of different isolation background on species or varieties level.2. By sequence analysis, two virulence-associate genes with important functions were filtered out. Moreover, the structure and function of these two proteins were influenced to some extent with the amino acid change. It is suggested that genes RAD23 and HST302 were related to the virulent difference between two groups of genotype strains.3. The strategies of nonsense SNPs and the InDels causing frameshift mutations show high-efficiency in sceening potential virulence-associated gene.4. Gene CNAG₀1032 was ultimately determined as a novel virulence-associated gene.5. Gene CNAG₀1032 mutant and its complemented strain were successfully constructed, which can be useful in further research of virulence and pathogenesis of Cryptococcus neoformance.