Open Access Research article

Variation in chromosome copy number influences the virulence of Cryptococcus neoformans and occurs in isolates from AIDS patients

Guanggan Hu1, Joyce Wang1, Jaehyuk Choi1, Won Hee Jung2, Iris Liu1, Anastasia P Litvintseva3, Tihana Bicanic4, Rajeev Aurora5, Thomas G Mitchell3, John R Perfect6 and James W Kronstad1*

Author Affiliations

1 The Michael Smith Laboratories, Department of Microbiology and Immunology, and Faculty of Land and Food Systems, University of British Columbia, Vancouver, B.C., V6T 1Z4, Canada

2 Department of Biotechnology, Chung-Ang University, Anseong-Si, Gyeonggi-Do, 456-756, Republic of Korea

3 Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, North Carolina, USA

4 Research Centre of Infection and Immunity, St. George's University of London, London, UK

5 Department of Molecular Microbiology and Immunology, Saint Louis University School of Medicine, St. Louis, Missouri, USA

6 Department of Medicine, Division of Infectious Diseases and International Health, Duke University Medical Center, Durham, North Carolina, USA

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BMC Genomics 2011, 12:526  doi:10.1186/1471-2164-12-526

Published: 27 October 2011

Additional files

Additional file 1:

Genes up-regulated in the black strain B4 (Table S1). A microarray comparison of the transcriptomes of the black B4 isolate and the white W2 isolate identified genes with greater than two-fold elevated transcript levels in the B4 isolate.

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Additional file 2:

Genes up-regulated in the white strain W2 (Table S2). A microarray comparison of the transcriptomes of the black B4 isolate and the white W2 isolate identified genes with greater than two-fold elevated transcript levels in the W2 isolate.

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Additional file 3:

Comparison of gene expression for a second-generation black strain (Figure S1). Gene expression was compared for the CBS7779-B4 strain and the "second generation" black strain CBS7779-W2BA by microarray analysis. Strain CBS7779-W2BA was obtained from the white strain CBS7779-W2 (Figure 1). Two arrays were employed and two biological repeats were performed to examine transcript levels. Columns 1 and 2 each represent a microarray experiment and each row represents the expression of a gene on the array arranged by its chromosomal position. The relative expression levels are represented by color as shown in the bar.

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Additional file 4:

CGH analysis of additional strains with chromosome copy number variation (Figure S2). Passage of the white variant CBS7779-W2 (disomic for chr 13) in culture and analysis of black or white isolates by CGH revealed changes at additional chromosomes. The black variant W2-BB showed copy number increase for a segment of chr 12, and the white isolate W2-WB gained a segmental changes for chr 12 and chr 14. The black variant strain CBS7779-B1 was confirmed to be monosomic by CGH and additional variants were identified in culture (Figure 6). In addition, white variants of strain B1 were subsequently screened for black variants (B1-WC-B1, disomic for chr 13; B1-WC-B4, monosomic for all chromosomes).

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Additional file 5:

CGH analysis of additional strains obtained from mice (Figure S3). Passage of the CBS7779 variants W2, W3 and B6 in mice lead to variation at chr 4 and chr 13. As described in the text and presented in Figure 6, white and black strains collected from the lungs and brains of mice infected with CBS7779 variants were compared to the reference genome of strain H99 by CGH.

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Additional file 6:

Phenotypic characterization of variants of the laboratory strain H99 (Figure S4). (A) The screen for variants with reduced melanin production is shown for tagged strains on L-DOPA medium containing neomycin. This procedure yielded the strains analyzed in Figure 7. (B) The capsule size and cell morphology of the variants were examined with india ink staining. This size bar is 10 μm. (C) Summary of the phenotypes of each variant.

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Additional file 7:

Strain list (Table S3). The strains employed in the study are listed in Table S3.

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Additional file 8:

CGH analysis of 18 clinical and environmental strains (Figure S5). The strains were analyzed along with strains A5-35-17 and JP1086 (Figure 8) using the array for the reference strain H99.

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Additional file 9:

CGH analysis of isolates from HIV/AIDS patients (Figure S6). Three isolated colonies were tested for patients HC-2, HC-3 and HC-5, and a representative colony is shown for the three colonies from patient HC-6. The analysis of another HC-6 colony is shown in Figure 8 (all three colonies had the same chromosome complement).

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Additional file 10:

Analysis of three HC-6 isolates by fluorescence-activated flow cytometry (Figure S7). The ploidy of the three isolates from patient HC-6 were examined and the haploid strain H99 and the diploid strain KW5 were included as controls.

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Additional file 11:

Primer list for strain construction (Table S4). The primers employed to insert a selectable marker on chr 13 of strain H99 are listed in Table S4.

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Additional file 12:

Primer list for qPCR (Table S5). The primers employed for quantitative real time PCR to confirm the copy number of chromosomes 4 and 13 are listed in Table S5.

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