Abstract
BackgroundThere are very few molecular genetic tools available to study the apicomplexan parasite Cryptosporidium parvum. The organism is not amenable to continuous in vitro cultivation or transfection, and purification of intracellular developmental stages in sufficient numbers for most downstream molecular applications is difficult and expensive since animal hosts are required. As such, very little is known about gene regulation in C. parvum.ResultsWe have clustered whole-genome gene expression profiles generated from a previous study of seven post-infection time points of 3,281 genes to identify genes that show similar expression patterns throughout the first 72 hours of in vitro epithelial cell culture. We used the algorithms MEME, AlignACE and FIRE to identify conserved, overrepresented DNA motifs in the upstream promoter region of genes with similar expression profiles. The most overrepresented motifs were E2F (5′-TGGCGCCA-3′); G-box (5′-G.GGGG-3′); a well-documented ApiAP2 binding motif (5′-TGCAT-3′), and an unknown motif (5′-[A/C] AACTA-3′). We generated a recombinant C. parvum DNA-binding protein domain from a putative ApiAP2 transcription factor [CryptoDB: cgd8_810] and determined its binding specificity using protein-binding microarrays. We demonstrate that cgd8_810 can putatively bind the overrepresented G-box motif, implicating this ApiAP2 in the regulation of many gene clusters.ConclusionSeveral DNA motifs were identified in the upstream sequences of gene clusters that might serve as potential cis-regulatory elements. These motifs, in concert with protein DNA binding site data, establish for the first time the beginnings of a global C. parvum gene regulatory map that will contribute to our understanding of the development of this zoonotic parasite.
Highlights
There are very few molecular genetic tools available to study the apicomplexan parasite Cryptosporidium parvum
There is a cascade of tightly regulated expression across the 72-hour intracellular life cycle of C. parvum
Most clustering methods are restricted to a one-to-one mapping scheme where one gene is assigned to only a single cluster, known as hard clustering (examples are k-means, Self Organizing Maps (SOM) and hierarchical clustering), while soft clustering can assign genes with a metrics value indicating the strength of its association with a cluster
Summary
There are very few molecular genetic tools available to study the apicomplexan parasite Cryptosporidium parvum. The organism is not amenable to continuous in vitro cultivation or transfection, and purification of intracellular developmental stages in sufficient numbers for most downstream molecular applications is difficult and expensive since animal hosts are required. Each parasite is 5 μm or smaller (depending on lifecycle stage; [6]) in a host cell that is a hundred to thousand times larger in volume. Given these complications of size, the post-infection parasite cannot be isolated from host cells in sufficient numbers, nor can sufficient post-infection parasite protein or RNA be obtained for most downstream molecular applications. C. parvum is not amenable to continuous in vitro cultivation or genetic dissection [7,8]
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