The Cryptococcus neoformans transcriptome at the site of human meningitis


Cryptococcus neoformans is the leading cause of fungal meningitis worldwide. Previous studies have characterized the cryptococcal transcriptome under various stress conditions, but a comprehensive profile of the C. neoformans transcriptome in the human host has not been attempted. Here, we extracted RNA from yeast cells taken directly from the cerebrospinal fluid (CSF) of two AIDS patients with cryptococcal meningitis prior to antifungal therapy. The patients were infected with strains of C. neoformans var. grubii of molecular type VNI and VNII. Using RNA-seq, we compared the transcriptional profiles of these strains under three environmental conditions (in vivo CSF, ex vivo CSF, and yeast extract-peptone-dextrose [YPD]). Although we identified a number of differentially expressed genes, single nucleotide variants, and novel genes that were unique to each strain, the overall expression patterns of the two strains were similar under the same environmental conditions. Specifically, yeast cells obtained directly from each patient's CSF were more metabolically active than cells that were incubated ex vivo in CSF. Compared with growth in YPD, some genes were identified as significantly upregulated in both in vivo and ex vivo CSF, and they were associated with genes previously recognized for contributing to pathogenicity. For example, genes with known stress response functions, such as RIM101, ENA1, and CFO1, were regulated similarly in the two clinical strains. Conversely, many genes that were differentially regulated between the two strains appeared to be transporters. These findings establish a platform for further studies of how this yeast survives and produces disease. © 2014 Chen et al.






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Chen, Yuan, Dena L Toffaletti, Jennifer L Tenor, Anastasia P Litvintseva, Charles Fang, Thomas G Mitchell, Tami R McDonald, Kirsten Nielsen, et al. (2014). The Cryptococcus neoformans transcriptome at the site of human meningitis. mBio, 5(1). 10.1128/mBio.01087-13 Retrieved from

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Dena L. Toffaletti

Associate Professor in Medicine

As an investigator in John Perfect's laboratory, I have continued to study the molecular pathogenesis of Cryptococcus neoformans. We are interested in identifying virulence genes expressed by the fungus during infection using our rabbit model of cryptococcal meningitis and during in vitro exposure to the pathological temperature of 37 degrees C. By PCR differential display, we are identifying genes associated with pathogenesis of the organism and assessing their effects at the molecular level using a 2-hybrid system. In addition, we are disrupting targeted virulence genes using biolistic transformation and assessing their role in the virulence of C neoformans in rabbit and murine models of cryptococcal disease.

Key words: Cryptococcus neoformans
Molecular Pathogenesis


Thomas Greenfield Mitchell

Associate Professor Emeritus in Molecular Genetics and Microbiology

Among patients with AIDS, leukemia or other cancers, organ or bone marrow transplants, and similar immunocompromising risk factors, the incidence of opportunistic mycoses and the number of different fungal pathogens are increasing dramatically. For many of these fungi, the definition of a species and the recognition of pathogen are highly problematic. Conventional methods of identification are based on morphological and physiological characteristics and are often time-consuming, difficult to interpret, and inconsistent. This laboratory is using DNA-based methods to (i) identify fungal pathogens, (ii) resolve taxonomic issues, (iii) facilitate epidemiological studies, (iv) recognize strains with clinically relevant phenotypes, such as resistance to antifungal drugs, (v) elucidate the origin(s) of diversity and the population genetics of the major pathogens, and (vi) explore functional genomics to identify virulence factors. We have developed reliable methods to genotype strains and are analyzing gene sequences to clarify the phylogeny of controversial taxa.

To conduct rigorous population studies of Candida albicans, we developed single-locus markers based on polymorphisms of PCR products. Genotypic frequencies and segregation patterns at these loci have confirmed that C. albicans is diploid and suggest that some form of recombination occurs in this "asexual" yeast. To investigate whether separate populations of C. albicans exist in disparate geographical locations, we compared strains collected from healthy and HIV-infected persons in U.S. and Brazil. Although a number of different genotypes were recognized at each location, the same multilocus genotype was prevalent among the clinical isolates, indicating a remarkable homogeneity among these populations.

We are using DNA-based methods to compare global isolates of Cryptococcus neoformans from patients with AIDS and other sources, to analyze the distribution and relatedness of strains, to identify genotypes of clinical importance, and to create linkage map of this pathogen. To determine the source of C. neoformans in patients, we developed a genetic markers to investigate the structure of clinical and environmental populations. With analysis of quantitative trait loci, specific genotypes will be identified that represent clones that have significantly diverged with respect to clinically relevant phenotypes, including susceptibility to antifungal drugs and the expression of virulence factors. We are investigating genomic evolution and phenotypic variation in natural populations of C. neoformans. These approaches will correlate genotypes with pathobiological phenotypes, leading to beneficial and predictive information about the epidemiology, diagnosis and prognosis of cryptococcosis in patients with AIDS.

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