Glycolysis Related Transcripts.

<p>The relative expression using 2-72 hr data is graphed for the annotated enzymes involved in glycolysis.</p

Functional Categories Distribution across 2-24 hr.

<p>Functional Categories Distribution across 2-24 hr.</p

The <em>Cryptosporidium Parvum</em> Transcriptome during In Vitro Development

<div><p>Cryptosporidiosis is caused by an obligate intracellular parasite that has eluded global transcriptional or proteomic analysis of the intracellular developmental stages. The transcript abundance for 3,302 genes (87%) of the <em>Cryptosporidium parvum</em> protein coding genome was elucidated over a 72 hr infection within HCT8 cells using Real Time-PCR. The parasite had detectable transcription of all genes <em>in vitro</em> within at least one time point tested, and adjacent genes were not co-regulated. Five genes were not detected within the first 24 hr of infection, one containing two AP2 domains. The fewest genes detected were at 2 hr post infection, while 30% (985) of the genes have their highest expression at 48 and/or 72 hr. Nine expression clusters were formed over the entire 72 hr time course and indicate patterns of transcriptional increases at each of the 7 time points collected except 36 hr, including genes paralleling parasite 18S rRNA transcript levels. Clustering within only the first 24 hr of infection indicates spikes in expression at each of the 4 time points, a group paralleling 18S rRNA transcript levels, and a cluster with peaks at both 6 and 24 hr. All genes were classified into 18 functional categories, which were unequally distributed across clusters. Expression of metabolic, ribosomal and proteasome proteins did not parallel 18S rRNA levels indicating distinct biochemical profiles during developmental stage progression. Proteins involved in translation are over-represented at 6 hr, while structural proteins are over-represented at 12 hr. Standardization methods identified 107 genes with <80% at a single of its total expression at a single time point over 72 hr. This comprehensive transcriptome of the intracellular stages of <em>C. parvum</em> provides insight for understanding its complex development following parasitization of intestinal epithelial cells.</p> </div

<i>C. parvum</i> Transcriptome 2–24 hr Relative Expression Cluster Analysis.

<p>The DIANA algorithm was used to cluster each gene's relative expression using the absolute cosign distance metric. For the heatmap each gene is represented in one row and each column represents the percent expression at one time point. (<b>A</b>)The relative expression (%) was determined for each gene's median normalized value over the first 24 hr of the time course, which includes 4 time points (3297 genes). Six clusters (A–F) of genes are present with a dendrogram distance cutoff of 1, as depicted with the color bar to the right of the heatmap. (<b>B</b>) The average gene expression per cluster (+/− SD) using the first 4 time points (24 hr) is plotted with the number of genes found in each cluster within the ().</p

Genes with no Detectable Expression in the first 24 hr.

<p>Genes with no Detectable Expression in the first 24 hr.</p

Distribution of Genes Across Clusters.

<p>The percentage of each of 13 functional categories was determined across all clusters. Five categories were omitted: Apicomplexan, <i>Cryptosporidium</i>, Oxidative Stress, Hypothetical, and Conserved Hypothetical. Chi-square testing was used to determine any significance in the distribution of the number of genes across clusters using the null hypothesis that the functional classifications would be equally distributed across clusters. Data was using the 2–72 hr (A) and 2–24 hr (B) data sets. Chi-square significance: * = <0.05, ** = <0.01, *** = <0.001, **** = <0.0001.</p

Chromosome 3 Peptidase Loci are not Co-expressed.

<p>Twelve genes, including 11 peptidase genes are present in tandem on chromosome 3. (<b>A</b>) The protease motifs were identified and color coded based on the conservation level. (<b>B</b>) RT-PCR for each gene was normalized to <i>C. parvum</i> 18S rRNA and the median for four biological replicates for the 7 time point time course is shown. (<b>C</b>) The normalized expression was then averaged by each gene's total expression for all 7 time points.</p

Functional Categories Distribution across 2-72 hr.

<p>Functional Categories Distribution across 2-72 hr.</p

Unique Asexual Stage Gene Expression.

<p>Gene transcription fluctuates during the infection time course. Most genes are detected at all 4 time points within the first 24 hours of infection (asexual replication). Gene lists were constructed containing the gene IDs for all genes with detectable transcription (non-zero) at 2, 6, 12, and 24 hr time points. These lists were compared to show the number of genes with overlapping expression at all combination of the four time points used. Blue = 2 hr, yellow = 6 hr, green = 12 hr, and red = 24 hr <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031715#pone.0031715-Oliveros1" target="_blank">[72]</a>.</p

Quantification of Parasite Stage Progression.

<p>HCT-8 coverslips were infected and stained with pan-specific antibody to quantify <i>C. parvum</i> infection. >300 parasites were counted on 15–30 frames for each of four replicates for each time point. The average for each of the 3 stages of parasite determined was converted into a percentage of the total parasite detected per time point. zoite  =  sporozoite/merozoite, trophozoite  =  all trophozoites (size of stained parasite was > zoite but trophozoite.</p