Abstract
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 Cryptosporidium parvum 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 in vitro 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 C. parvum provides insight for understanding its complex development following parasitization of intestinal epithelial cells.
Highlights
Cryptosporidium species are global contaminants of surface water and are the second leading cause of human gastrointestinal illness in the United States
We used Real Time (RT)-PCR to describe the transcriptional profile of 3,302 C. parvum genes during a 72 hr in vitro infection time course using the epithelial cell line, HCT8, resulting in the first comprehensive transcriptome for this obligate intracellular parasite
Gene expression ranged from 10210 to 0.5 transcripts relative to C. parvum 18S rRNA, while the median expression relative to rRNA across the time course did not change (Table 1)
Summary
Cryptosporidium species are global contaminants of surface water and are the second leading cause of human gastrointestinal illness in the United States. Due to its resistance to standard water chlorine disinfection, Cryptosporidium is a public health concern and a potential water-borne bioterrorism agent due to its low infectious dose (as low as 10 oocysts) and its ability to be stably delivered to the human population en masse [3]. Illness varies from profuse, self-limiting diarrhea to life threatening malabsorption and dehydration depending on immune status. Effective therapeutics have not been formulated because the eukaryotic parasite has a condensed genome lacking many of the traditional drug targets [4]. Most of the remaining genes have remained functionally uncharacterized, thereby limiting pharmacological targets [5,6]
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