ABSTRACT
Objective To analyze the genomic sequences of Cryptococcus neoformans var grubii strains of two genotypes with different virulence and to screen out the virulence-associated genes. Methods A clinical strain (IFM56800) with the strongest virulence and an environmental strain (IFM56731) with the weakest virulence were screened out for whole genome sequencing analysis. The results of sequencing analy-sis were comprehensively analyzed by using the method of comparative genomics. Genetic variations were ex-tensively screened by using the strategies of non-synonymous single nucleotide polymorphisms ( nsSNPs), nonsense SNPs and the insertions or deletions ( InDels) causing frameshift mutations. The filtered genes were sequenced in 20 experimental strains. The whole RNAs were extracted and then the full-length cDNAs were sequenced by using the rapid amplification of 5′ and 3′ cDNA ends (RACE) method. Results By whole genome sequencing, valid data with high coverage (127 times and 111 times) was obtained in both the environmental strain IFM56731 and the clinical strain IFM56800. The data of InDels and SNPs were statisti-cally analyzed, respectively. Six genes were chosen for further analysis based on the strategies of nonsense SNPs and the InDels causing frameshift mutations. The six genes were amplified and sequenced in all of the experimental strains, three of which were further analyzed with cDNA sequencing. Ultimately, the location and structure of CNAG_01032 gene were determined. The predicted nonsense mutation locus was verified to present in the actual mRNA. Conclusion The strategies of nonsense SNPs and the InDels causing frame-shift mutations showed high-efficiency in screening potential virulence-associated genes. The CNAG_01032 gene was screened out as a novel virulence-associated gene.
ABSTRACT
Objective Cryptococcus neoformans var.grubii isolates from 6 different cities in our country using multilocus microsatellite typing(MLMT) method were genotyped to explore the genotypic distribution of the variety.Methods The DNA of forty-three isolates of Cryptococcus neoformans var.grubii was extracted.The DNA fragments covering microsatellite loci CNG1,CNG2 and CNG3 were amplified using PCR,and then sequenced.The numbers of each motif repeat in 3 microsatellite regions("TA" repeats for CNG1,"GA" repeats for CNG2,and "CAT" repeats for CNG3) were calculated.According to the repeat numbers of these motif,the MLMT types of 43 strains of Cryptococcus neoformans var.grubii were determined.Results Out of 43 isolates,the percentage of MLMT-17 was 83.72%.In the clinical and environmental isolates,the percentages of MLMT-17 were 86.67% and 70%,respectively.Two new genotypes MLMT-39 and-40 were found.Conclusion MLMT-17 of Cryptococcus neoformans var.grubii is prevalent in both clinical and community environment in China.Implying the most clinical strains which resulted in cryptococcosis originated from indigenous environmental strains.
ABSTRACT
Objective To understand the genotype distribution of clinical Cryptococcus neoformans var.grubii strains from China and Brazil using multilocus microsatellite typing(MLMT) method and to study the difference in their MLMT genotyping.Methods DNA was extracted from the identified 69 clinical Cryptococcus neoformans var.grubii strains.DNA fragments covering microsatellite loci(CNG1,CNG2,and CNG3) were amplified by PCR and sequenced.The number of each motif repeats in 3 microsatellite regions("TA","GA",and "CAT" repeats for CNG1,CNG2,and CNG3,respectively) was calculated.The MLMT types of 69 clinical Cryptococcus neoformans var.grubii strains were determined according to the repeat number of different motifs.Data were analyzed using SPSS 11.5 software.Results Five genotypes were identified in 33 clinical strains from China.Of these strains,29 were MLMT-17,accounting for 87.88% of the total strains.Ten genotypes were identified in 36 clinical strains from Brazil.Of the 36 strains,19 were MLMT-13,accounting for 52.78% of the total strains.Conclusion The difference is great in major genotype distributions of the clinical Cryptococcus neoformans var.grubii strains from China and Brazil.The genotype of clinical strains from Brazil is diversely distributed.