Population Genomics And Relevant Phenotype Analysis Of VNI Genotype C. Neoformans

ObjectivesCryptococcus neoformans(C.neoformans)is a kind of opportunistic pathogenic yeast with high mortality rate,and VNI genotype is the most important pathogenic genotype of C.neoformans.Majority of cryptococcosis in China(more than 90%)is caused by VNI genotype isolates of C.neoformans.Previous studies on the molecular epidemiology of C.neoformans in China were usually based on the sequence information of single or several gene loci,which could not accurately analyze the population structure,evolution relationship and phenotypic characteristics of related strains.In this study,we intend to study the population genetics and phenotype of Chinese and Asian VNI genotype C.neoformans on genomic level for the first time.The first part is to study the genomic typing and population genetic characteristics of VNI genotype isolates of C.neoformans collected from China;the second part is to compare the differences between Chinese VNI genotype clinical strain population and environmental strain population from the perspective of population genomics;the third part is to explore the population structure and evolution relationship of the whole Asian VNI genotype C.neoformans by using the global VNI genotype genomic data.In the fourth part,the phenotypic characteristics of VNI C.neoformans were explored and the target gene set possibly related to the phenotype was found through the phenotypic experiments of C.neoformans growth curve,cell wall fluorescence intensity and heteroresistance as well as GWAS analysis.MethodsPart I The clinical strains(n=26)and environmental isolates(n=23)of VNI genotype C.neoformans were randomly collected from different provinces of China;PE library construction and "Next-generation" sequencing(NGS)of whole genome was carried out for C.neoformans;genome typing was carried out for VNI genotype C.neoformans in China by phylogenetic analysis and genomic principal component analysis,and the typing results were compared with the previous MLST typing results based on the 7 loci;Tajima's D value and Fst value were respectively used to analyze the population genetics of VNI genotype C.neoformans in China.Part II SNP density and CLR positive selection analysis were performed on clinical and environmental strains of VNI genotype C.neoformans in China respectively,and genomic regions with different traits were extracted.The genes contained in those genomic regions were extracted,and their functions were annotated,and the GO enrichment analysis was further performed.Virulence gene bank of VNI genotype C.neoformans was also established.Part III Principal component analysis based on SNP data and STRUCTURE analysis of VNI genotype C.neoformans in Asia were carried out;the CLR positive selection test was carried out for each group of VNI genotype C.neoformans in Asia,and the functional annotation and GO pathway enrichment analysis were carried out for the target gene set obtained from the analysis;the evolution relationship between each group of VNI genotype C.neoformans in Asia was inferred by Tree Mix software;The origin time of three Asian VNI C.neoformans group was calculated by Beast software;The gene introgression analysis was carried out by Popgenome software;Copy number variation was analyzed by GATK software.Part IV The 25? growth curve phenotype of 86 strains of VNI C.neoformans was tested;The cell wall CFW fluorescence intensity of 75 strains of VNI C.neoformans was tested;The medium fluconazole(32 mg / L)heteroresistance phenotype of 109 strains of VNI C.neoformans was tested;The high fluconazole heteroresistance(64 mg / L)phenotype of 101 strains of VNI was tested.In addition,we used genome microevolution and transcriptome analysis to further study the heteroresistance phenotype of C.neoformans.GWAS analysis was performed on the above phenotype experimental resultsResultsPart I In China,most of the patients infected by VNI C.neoformans were HIV negative(96.15%,25 / 26).Chinese VNI C.neoformans population is mainly divided into three groups: China1,China2 and China3.China1 is the dominant group of VNI C.neoformans in China,accounting for 81% of clinical strains and 61% of environmental strains.The results of whole genome typing of VNI C.neoformans are different from those of MLST.ST359,ST360,ST226 and ST5 belong to China1 populatio.ST31 belongs to China2 and China3 population under genome typing,which suggests that the whole genome typing has the ability to distinguish population genetic diversity more precisely The results of Tajima's D analysis indicated that the positive selection effect of China1 group was much stronger than that of the other two groups.Part II Compared with the environmental strains,there are 20 unique high SNP density genomic regions in Chinese clinical strains of VNI C.neoformans.These regions contain 105 genes and are enriched in the pathway related to the synthesis of cell wall capsule(GO: 0030117).Among these 105 genes,there are genes encoding ?-glucosidase,which are related to the reconstruction of C.neoformans cell wall capsule.The results of CLR positive selection analysis indicate that China's VNI clinical strains have 43 highly positive selection regions compared with the environmental strains.These regions contain genes related to the regulation of chitin and chitosan content in the cell wall of C.neoformans and are related to the reconstruction of the cell wall capsule of C.neoformans.Part III The results of PCA analysis based on SNP data indicated that VNI C.neoformans in Asia could be divided into three main groups: Asia1,Asia2 and Asia3.All the strains of China1,which are dominant in China VNI C.neoformans,belong to Asia1 group.The results showed that the Asia1 group was the dominant group in cryptococcal infection of HIV negative patients(73.2%,71 / 97).The results of Tree Mix analysis suggest that the Asia1 population is in the newly occurred position and the Asia3 population is in the more ancestral position.According to Beast's prediction,Asia1 population originated about 2100 years ago,and Asia3 population about 140000 years ago.The detection results of whole genome CLR positive selection indicate that Asia1 population has 40 unique highly positive selection regions compared with Asia2 and Asia3 population.These regions contain genes related to the regulation of capsular polysaccharide of C.neoformans,and these genes are in the gene intreogression peak region from Asia3 population to Asia1 population.It was found that Asia1 populations had 21 genes with copy number variations compared with Asia2 and asia3 populations.Part IV The CFW fluorescence intensity of VNI C.neoformans in the temperate and subtropical zone of Asia was significantly higher than that in the tropical zone of Asia(P = 1.832e-05 < 0.01);the growth rate at 25? of VNI C.neoformans in the mild and subtropical zone of Asia was significantly faster than that in the tropical zone of Asia(P = 1.289e-06< The results of Spearman correlation analysis showed that CFW fluorescence intensity was positively correlated with the growth rate at 25?(p = 0.034 < 0.05,R = 0.25);GWAS analysis revealed six genes that might be related to the growth rate at 25?.GWAS analysis found four genes that may be related to the heteroresistance phenotype of fluconazole;four of the 33 non synonymous SNPs detected by genome micro evolution experiment fell on the related genes found by GWAS analysis;transcriptome analysis found 10 differentially expressed genes related to heteroresistance,including the gene encoding alcohol dehydrogenase,while alcohol dehydrogenase has been proved to be related to Candida yeast's resistance to fluconazoleConclusionsPart I VNI isolates of C.neoformans in China can be divided into three groups according to the genome typing,among which the “China1” group is the dominant and has undergone strongpositive selections.ST5 strain belongs to China1 population under genome typing.Part II The specific ability of Chinese clinical strains of VNI C.neoformans to infect the immunocompetent population may be related to the remodelling of C.neoformans cell wall and capsule,and the genes related to ?-glucosidase and chitinase may be the possible target gene set.Part III All the strains of China1 group belong to Asia1 group under genome typing of Asian VNI C.neoformans.The predominance of Asia1 group in HIV negative patients may be related to the remodeling of C.neoformans cell wall and capsule,and the gene related to ?-glucosidase may be the possible target.Part IV The results of cell wall CFW fluorescence intensity and 25? growth curve phenotype experiment showed that the difference in the ability of Asian VNI C.neoformans to infect immunocompetnet patients might be related to the change of C.neoformans cell wall components and the adaptation to low temperature environment.It is found that the heteroresistance phenotype of VNI C.neoformans to fluconazole is closely related to the genes related to chitin regulation and the genes encoding alcohol dehydrogenase,which will provide a new idea for the treatment of infection caused by fluconazole resistant VNI C.neoformans.