Transcriptome analysis reveals unique metabolic features in the Cryptosporidium parvum Oocysts associated with environmental survival and stresses

Abstract

Background: Cryptosporidium parvum is a globally distributed zoonotic parasite and an important opportunistic pathogen in immunocompromised patients. Little is known on the metabolic dynamics of the parasite, and study is hampered by the lack of molecular and genetic tools. Here we report the development of the first Agilent microarray for C. parvum (CpArray15K) that covers all predicted ORFs in the parasite genome. Global transcriptome analysis using CpArray15K coupled with real-time qRT-PCR uncovered a number of unique metabolic features in oocysts, the infectious and environmental stage of the parasite.

Results: Oocyst stage parasites were found to be highly active in protein synthesis, based on the high transcript levels of genes associated with ribosome biogenesis, transcription and translation. The proteasome and ubiquitin associated components were also highly active, implying that oocysts might employ protein degradation pathways to recycle amino acids in order to overcome the inability to synthesize amino acids de novo. Energy metabolism in oocysts was featured by the highest level of expression of lactate dehydrogenase (LDH) gene. We also studied parasite responses to UV-irradiation, and observed complex and dynamic regulations of gene expression. Notable changes included increased transcript levels of genes involved in DNA repair and intracellular trafficking. Among the stress-related genes, TCP-1 family members and some thioredoxin-associated genes appear to play more important roles in the recovery of UV-induced damages in the oocysts. Our observations also suggest that UV irradiation of oocysts results in increased activities in cytoskeletal rearrangement and intracellular membrane trafficking.

Conclusions: CpArray15K is the first microarray chip developed for C. parvum, which provides the Cryptosporidium research community a needed tool to study the parasite transcriptome and functional genomics. CpArray15K has been successfully used in profiling the gene expressions in the parasite oocysts as well as their responses to UV-irradiation. These observations shed light on how the parasite oocysts might adapt and respond to the hostile external environment and associated stress such as UV irradiation.

Figure 1

Correlation plots of signal intensities (means of normalized median signals) between Cy3 and Cy5 dye swaps (R²=0.9879) (A) and biological replicates (R²=0.9886) (B). In panel B, data for replicates 1 and 2 were derived from 0.5 h and 5 h groups, each including two untreated controls and two UV-irradiation samples (oocysts) that were allowed to recover for 0.5 h or 5 h, respectively.

Figure 2

Evaluation of inter-array variations by plotting of the mean signal intensities with standard deviations (SDs) of all individual probes among all 8 microarrays, and inter-probe variations by plotting the means and SDs of all individual genes from multiple probes (inset).

Figure 3

Validation of microarray data by real-time qRT-PCR for selected C. parvum genes. (A) Relative mRNA levels of C. parvum genes in the oocyst stage. Microarray signals are shown as the means of normalized signals derived from all probes in all 8 arrays (left axis). Levels determined by semi-qRT-PCR are displayed as the fold differences in relative to the mean of all samples in at least three biological replicates (right axis). (B) Fold changes of the gene expressions in C. parvum oocysts recovered for 30 min after UV-treatment. Bars indicate standard deviations (SDs).

Figure 4

Features of expressed genes in the C. parvum oocysts (Group I genes) as determined by microarray analysis. (A) Categorization of expressed Group I genes by major functional groups. (B) Functional categorization of Group I genes by expression levels (by quartiles) in comparison with the lowly or unexpressed Group II genes.

Figure 5

Illustration of the expression levels of enzymes within the glycolytic pathway and major connections in the C. parvum oocysts as determined by microarray analysis. The expression levels are grouped into 5 major groups by color and size. Abbreviations: ACC, acetyl-CoA carboxylase; AceACL, acetic acid-CoA ligase (aka acetyl-CoA synthetase); ACL, fatty acid-CoA ligase (aka acyl-CoA synthetase); ADH, alcohol dehydrogenase; ADH-E, type E alcohol dehydrogenase (bifunctional); FAS1, type I fatty acid synthase; GAPDH, glyceraldehyde phosphate dehydrogenase; GBE, glycogen branching enzyme; GDBE, glycogen debranching enzyme; GDH, glycerol phosphate dyhydrogenase; HK, hexokinase; LCE, long chain fatty acyl elongase; LDH, lactate dehydrogenase; MDH, malate dehydrogenase; ME, malic-enzyme; PDC, pyruvate decarboxylase; PEPCL, phosphoenolpyruvate carboxylase; PFK, phosphofructokinase; PGI, phosphoglucose isomerase; PGK, phosphoglycerate kinase; PGM, phosphoglycerate mutase; PGluM, phosphoglucose mutase; PK, pyruvate kinase; PKS1, type I polyketide synthase; PNO, pyruvate:NADP+ oxidoreductase; T6PS-TP, trehalose 6-phosphate synthase; TIM, triosephosphate isomerase; UGGP, UDP-galactose/glucose pyrophosphorylase.

Figure 6

Relative level of CpLDH1 gene expression in the oocysts, excystated sporozoites and various intracellular developmental stages in comparison with selected genes as determined by qRT-PCR. (A) Comparison of CpLDH1 expression with 4 other glycolytic genes. All levels are relative to that of CpLDH1 in the oocysts. (B) Comparison of CpLDH1 expression with three other genes responsible for producing different organic end products. In this panel, individual genes were separately calibrated, relative to the highest level within individual genes.

Figure 7

Effect of UV₂₅₄ irradiation on the C. parvum oocyst viability as determined by a qRT-PCR-based in vitro infection assay. The ID₂₀ value was determined at 2.8 mJ/cm².