Pathway annotation by DAVID bioinformatics resource.

<p>The 65 significant genes (133 overlapped probes) which were differentially regulated in H99 compared to environmental strains were analyzed. Shown were representative of each annotation cluster detected. Count represents number of genes which match the pathway database, and % represents the percentage of gene hits among the total genes in the pathway database. Enrichment score (ES) of each group was measured by the geometric mean of the EASE Scores (modified Fisher Exact) associated with the enriched annotation terms that belong to this gene group. Population hit (Pop Hits) represents how many have the function name in your gene list of interest, and population total (Pop Total) represents how many genes in overall population has that function name in the background genome (all genes in the species of interest in DAVID database). False discovery rate (FDR) represents the percentages of test which might be false positive. <i>P</i> values were analyzed using Fisher exact score to identify which sub-populations are over- or under-represented in a sample. Data were considered significant if *<i>P</i><0.05.</p

Genome-Wide Transcription Study of <i>Cryptococcus neoformans</i> H99 Clinical Strain versus Environmental Strains

<div><p>The infection of <i>Cryptococcus neoformans</i> is acquired through the inhalation of desiccated yeast cells and basidiospores originated from the environment, particularly from bird’s droppings and decaying wood. Three environmental strains of <i>C</i>. <i>neoformans</i> originated from bird droppings (H4, S48B and S68B) and <i>C</i>. <i>neoformans</i> reference clinical strain (H99) were used for intranasal infection in C57BL/6 mice. We showed that the H99 strain demonstrated higher virulence compared to H4, S48B and S68B strains. To examine if gene expression contributed to the different degree of virulence among these strains, a genome-wide microarray study was performed to inspect the transcriptomic profiles of all four strains. Our results revealed that out of 7,419 genes (22,257 probes) examined, 65 genes were significantly up-or down-regulated in H99 versus H4, S48B and S68B strains. The up-regulated genes in H99 strain include <i>Hydroxymethylglutaryl-CoA synthase</i> (<i>MVA1</i>), <i>Mitochondrial matrix factor 1</i> (<i>MMF1</i>), <i>Bud-site-selection protein 8</i> (<i>BUD8</i>), <i>High affinity glucose transporter 3</i> (<i>SNF3</i>) and <i>Rho GTPase-activating protein 2</i> (<i>RGA2</i>). Pathway annotation using DAVID bioinformatics resource showed that metal ion binding and sugar transmembrane transporter activity pathways were highly expressed in the H99 strain. We suggest that the genes and pathways identified may possibly play crucial roles in the fungal pathogenesis.</p></div

Pathway analysis.

<p>Histogram shows a summary of DAVID pathway annotation analysis. The 65 genes which were commonly regulated in H99 versus three environmental strains were selected and analyzed. The number of significant genes and their percentages of genes in the database are as indicated.</p

Microarray analysis.

<p>(A) Pairwise correlation matrix. Four <i>C</i>. <i>neoformans</i> strains (H99, H4, S48B and S68B) were compared. Two independent samples were prepared for each fungal strain. Rep1–2: replicates of samples. Numbers in the box represents correlation coefficient values among groups. Dark box: high correlation; light box: low correlation. (B) Gene expression heat map. Dendrogram represents the colour-coded expression levels of the significantly regulated genes. The groups of genes which showed differential expressions in H99 versus other strains were as indicated. Low levels in H99 category indicates the genes with low expression levels in H99 (green) but high expression in the environmental strains (red). In contrast, high levels in H99 category indicates the genes with high expression levels in H99 (red) but show low expression in the environmental strains (green). Hierachrical cluster was performed with Euclidean distance metric and Ward’s Linkage rule clustering. Colour range represents log₁₀ (FC) of the microarray intensities.</p

Gene expression of H99 versus environmental strains.

<p>(A) Scatter plots show the expressions of total probes in the H99 versus H4, S48B or S68B. X and Y axis show log₁₀ raw intensity values. (B) Venn diagram. Significantly regulated probes (FC>2 or FC<-2, P<0.05) were selected from the comparisons of H99 versus each environmental strain. Venn diagram shows the distribution and the number of the genes (probes) in each fraction.</p

qRT-PCR verification.

<p>RNAs were extracted from H99, H4, S48B and S68B for qRT-PCR analysis. Two independent samples were prepared for each fungal strain. All samples were run in triplicates and data were shown as mean ± SD. Group statistical significance measured was by one-way ANOVA analysis (*<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001).</p

65 significant up- and down-regulated genes in <i>C</i>. <i>neoformans</i> H99 relative to non-virulent strains.

<p>^#No hit were obtained using <i>C</i>. <i>neoformans</i> mRNA sequence in NCBI blast database and a closest hit from <i>Saccharomyces cerevisiae</i> was selected. Only known genes or genes which matched blast database are described. vs: versus. The <i>P</i> value is determined by an ANOVA analysis, which compares among all sample pairs.</p

Different traits of <i>C</i>. <i>neoformans</i> strains.

<p>(A) Growth curve. <i>C</i>. <i>neoformans</i> strains (H99, H4, S48B and S68B) cells were cultured in triplicates at 37°C with gentle shaking. OD reading for each sample was measured throughout a time course of 108 hours. (B) E-test. Fungal strains were inoculated evenly onto SDA plate. An AMB or FLU-containing MIC strip was then placed on each plate and incubated at 37°C for 48 hours. Eclipse sizes which intersected with the MIC strips were recorded. Shown were mean±SD from 2 duplicate plates. Data were representative of two independent experiments. (C) Capsule formation. Fungal cells were incubated in PBS with or without the presence of 10% serum, and incubated for 24 hours at 37°C. Cells were then applied on glass slide for India ink staining.</p

<i>In vivo</i> virulence study among different <i>C</i>. <i>neoformans</i> strains.

<p>(A) Kaplan-Meier survival curve. C57BL/6 mice at age 8–12 weeks old were administrated intranasally with 2 × 10⁵ yeast cell suspension. Mice were infected with <i>C</i>. <i>neoformans</i> clinical strain H99 or environmental strains H4, S48B and S68B, and observed for a period of 42 days. (n = 6–9). (B and C) CFU assay. Lung (B) or brain (C) homogenates from <i>C</i>. <i>neoformans</i>-infected mice (n = 4) were serially diluted and plated onto agar plates in duplicates. Numbers of colonies (CFU/ml) were counted after 48 hours. CFU formations on the plates were measured and shown as mean ± SD. Group statistical significance measured was by two-way ANOVA analysis (*<i>P</i><0.05).</p

Primer sequences for qRT-PCR analysis.

<p>List of the forward and reverse primer sequences (5’-3’) of the up- and down-regulated genes selected for qRT-PCR analysis.</p