The Cryptococcus neoformans transcriptome at the site of human meningitis

Abstract

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.

Importance: Cryptococcus neoformans, an environmental, opportunistic yeast, is annually responsible for an estimated million cases of meningitis and over 600,000 deaths, mostly among HIV-infected patients in sub-Saharan Africa and Asia. Using RNA-seq, we analyzed the gene expression of two strains of C. neoformans obtained from the cerebrospinal fluid (CSF) of infected patients, thus creating a comprehensive snapshot of the yeasts' genetic responses within the human body. By comparing the gene expression of each clinical strain under three conditions (in vivo CSF, ex vivo CSF, and laboratory culture), we identified genes and pathways that were uniquely regulated by exposure to CSF and likely crucial for the survival of C. neoformans in the central nervous system. Further analyses revealed genetic diversity between the strains, providing evidence for cryptococcal evolution and strain specificity. This ability to characterize transcription in vivo enables the elucidation of specific genetic responses that promote disease production and progression.

FIG 1

(A) Hierarchical cluster analysis of gene expression based on log ratio of normalized read count per gene. The cluster displays expression patterns for six RNA-seq samples. The color key represents log₂-normalized transformed counts. (B) Number of genes that are significantly up/downregulated (FDR < 0.2) between two different conditions in both strains. Pairwise comparisons were made between three conditions: ex vivo CSF versus YPD, in vivo CSF versus YPD, and in vivo CSF versus ex vivo CSF. The numerical designation indicates the number of genes that were up/downregulated in the first condition compared to that in the second condition. See Table S2 in the supplemental material for a list of these genes.

FIG 2

Heatmaps showing the expression data of 20 genes that were significantly upregulated in ex vivo CSF and in vivo CSF compared to YPD in both strains (A) and the expression data of six genes that were significantly upregulated in in vivo CSF compared to in ex vivo CSF and YPD in both strains (B). (C) Hierarchical cluster analysis of the 100 most divergently expressed genes between G0 and HC1.

FIG 3

The proportions of the novel genes of G0 (A) and HC1 (B) that mapped to different species. (C) GO classification of novel genes of G0 and HC1 compared to those of H99. Percentages of genes annotated with certain GO terms compared to total number of annotated genes are shown in the figure. A hypergeometric test was used to test the enrichment of G0 and HC1 novel genes compared with H99 gene composition (*, P < 0.05; **, P < 0.01).