Microevolution of Serial Clinical Isolates of Cryptococcus neoformans var. grubii and C. gattii

Abstract

The pathogenic species of Cryptococcus are a major cause of mortality owing to severe infections in immunocompromised as well as immunocompetent individuals. Although antifungal treatment is usually effective, many patients relapse after treatment, and in such cases, comparative analyses of the genomes of incident and relapse isolates may reveal evidence of determinative, microevolutionary changes within the host. Here, we analyzed serial isolates cultured from cerebrospinal fluid specimens of 18 South African patients with recurrent cryptococcal meningitis. The time between collection of the incident isolates and collection of the relapse isolates ranged from 124 days to 290 days, and the analyses revealed that, during this period within the patients, the isolates underwent several genetic and phenotypic changes. Considering the vast genetic diversity of cryptococcal isolates in sub-Saharan Africa, it was not surprising to find that the relapse isolates had acquired different genetic and correlative phenotypic changes. They exhibited various mechanisms for enhancing virulence, such as growth at 39°C, adaptation to stress, and capsule production; a remarkable amplification of ERG11 at the native and unlinked locus may provide stable resistance to fluconazole. Our data provide a deeper understanding of the microevolution of Cryptococcus species under pressure from antifungal chemotherapy and host immune responses. This investigation clearly suggests a promising strategy to identify novel targets for improved diagnosis, therapy, and prognosis.IMPORTANCE Opportunistic infections caused by species of the pathogenic yeast Cryptococcus lead to chronic meningoencephalitis and continue to ravage thousands of patients with HIV/AIDS. Despite receiving antifungal treatment, over 10% of patients develop recurrent disease. In this study, we collected isolates of Cryptococcus from cerebrospinal fluid specimens of 18 patients at the time of their diagnosis and when they relapsed several months later. We then sequenced and compared the genomic DNAs of each pair of initial and relapse isolates. We also tested the isolates for several key properties related to cryptococcal virulence as well as for their susceptibility to the antifungal drug fluconazole. These analyses revealed that the relapsing isolates manifested multiple genetic and chromosomal changes that affected a variety of genes implicated in the pathogenicity of Cryptococcus or resistance to fluconazole. This application of comparative genomics to serial clinical isolates provides a blueprint for identifying the mechanisms whereby pathogenic microbes adapt within patients to prolong disease.

FIG 1

Aneuploid regions of all C. neoformans var. grubii isolates. For each sequenced isolate, the normalized read depth is shown in 5-kb windows along each chromosome relative to the H99 reference, and average normalized depth on the y axis is relative to ploidy levels.

FIG 2

Phylogenetic relationships of the serial isolates and comparison of duplicated genomic regions in six relapse isolates. (A) Phylogenetic tree inferred by using RAxML from 503,457 SNPs in the C. neoformans var. grubii genome (2.7% of the genome). A total of 1,000 bootstrap replicates were performed, and nodes with 100% bootstrap support are denoted by asterisks (*). (B) Copy number of three C. neoformans var. grubii chromosomes (chromosomes 2, 5, and 12) for six isolates with a large genomic duplication; orange regions show elevated copy numbers, and regions with normal copy number are shown in green.

FIG 3

Analyses of the highly amplified genomic sequences containing ERG11 in the isolates from patient 9. (A) The region of chromosome 1 (Chr1) that includes the ERG11 gene (CNAG_00040) was duplicated on Chr6. Two copies of this region on Chr1 from nucleotide 123675 to nucleotide 128150 were identified in the incident isolate (RSA-MW-1746), and the extra copy was mapped as an insertion into Chr6. A larger duplicated region was identified in the relapse isolate (RSA-MW-3615) with an average of 13× coverage on chromosome 1 from nucleotide 121438 to nucleotide 129598. (B) The TagMap assay was used to analyze the duplicated regions. Primers were designed within 200 bp of the break points. The two flanking regions were concatenated in reverse order (A2 → A1 and B2 → B1), and the sequencing reads were aligned to verify their tandem duplication. A large proportion of the reads could be aligned to the A2–A1 junction, indicating tandem alignment of the duplicated region in RSA-MW-3615. None of the reads aligned to the B2–B1 junction, indicating that no tandem arrangement of the duplicated region had occurred in RSA-MW-1746. These results provide more evidence that the other copy was inserted on Chr6. (C) Sequencing reads from the TagMap assay of RSA-MW-1746 aligned to Chr6 confirmed the insertion of an additional copy of this region from 991,188 to 991,395.

FIG 4

Survival curves for Galleria mellonella infected with incident and relapse isolates from patients 22 (A), 1 (B), 5 (C), and 15 (D). H99 (gray) and phosphate-buffered saline (PBS) (green) were used as positive and negative controls in the experiments. Fifteen larvae were infected per isolate, and each isolate was evaluated three times. Mantel-Cox tests showed significant differences (P < 0.01) in virulence between the incident (orange) and relapse (blue) isolates in these samples.

FIG 5

(A and B) Variations in capsule size were observed between the initial isolate, MW-RSA-1052 (A), and the relapse isolate, MW-RSA-3156 (B). Isolates were streaked onto YPD plates and grown for 3 days at 30°C, a condition that typically results in limited capsule production. An individual colony was resuspended in PBS containing India ink, subjected to brief vortex mixing, and examined at ×63 magnification. Bars, 20 µM. (C) The capsule radius for 50 cells was measured. The incident isolate had a much larger capsule size than the relapse isolate. P value, <0.0001. The cell body sizes were similar (data not shown).