Submitted to: Parasitology Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/10/2011
Publication Date: 3/5/2011
Citation: Jenkins, M.C., Obrien, C.N., Miska, K.B., Schwartz, R., Karns, J.S., Santin, M., Fayer, R. 2011. Gene expression during excystation of Cryptosporidium parvum oocysts. Parasitology Research. 109(2):509-513. Interpretive Summary: Cryptosporidiosis is an intestinal parasitic disease of humans and animals that is caused by the protozoa in the genus cryptosporidium. The two species known to infect humans are C. parvum and C. hominis; the former zoonotic because it can be transmitted between humans and animals, such as dairy calves. The disease generally begins by the ingestion of water or food contaminated with Cryptosporidium oocysts. The oocyst stage of the parasite is extremely well adapted to survival for long periods of time in the environment due to the presence of a tough outer coat composed of glycolipids and protein. After ingestion, four invasive stages, called sporozoites, are released from each oocyst in a process termed excystation. The purpose of the present study was to find out what genes are turned on during the excystation process, with the goal of identifying processes important to release of sporozoites. It was found that a wide range of genes are “up-regulated” in excysting C. parvum oocysts, but that the greatest up-regulation was in genes coding for structural proteins. These findings suggest that sporozoites may turn on genes that are important in host cell invasion and subsequent development in the host, and may point to processes that might be inhibited, and thereby prevent infection in the host.
Technical Abstract: The present study describes transcription of mRNA from genes encoding metabolic or structural proteins during excystation of Cryptosporidium parvum oocysts. RNA was harvested from C. parvum oocysts before excystation, and at 5, 10, and 15 min during excystation. Subtractive cDNA libraries were prepared by using mRNA from non-excysted C. parvum oocysts to “subtract out” mRNA from excysting oocysts. The “subtracted” cDNA was used to prepare libraries enriched for transcripts possibly involved in excystation. From these libraries, over 1000 expressed sequence tags (ESTs) were analyzed by DNA sequencing followed by BLAST-N and BLAST-X analysis. While several gene products involved in cell metabolism and cell signaling were consistently recovered, transcription levels, as reflected by the relative number of cDNA sequences, were highly up-regulated in genes coding for structural proteins such as Cp2, CpTSP, CpHC10, and CpSAg. Moreover, of the greater than 1000 clones analyzed, the highest number of ESTs detected in excysting oocysts were for hypothetical C. parvum proteins (CpHyP), whose function are presently unknown.