Cell Sorting-Assisted Microarray Profiling of Host Cell Response to Cryptosporidium parvum Infection

To study the transcriptional response of mammalian cells to infection with the intracellular apicomplexan parasite Cryptosporidium parvum, infected and uninfected cells were recovered from C. parvum-infected cell monolayers. This approach, which contrasts with a more conventional experimental design that compares infected to uninfected cell monolayers, enabled the identification of functional categories of genes that are differentially transcribed as a direct consequence of the presence of intracellular parasites. Among several categories of upregulated genes, glycoprotein metabolism was significantly overrepresented. To investigate whether these transcriptional changes affected the composition of the surface of infected cells, cells were probed with fluorescently labeled lectins. Among a panel of seven lectins, soybean agglutinin, which recognizes N-acetyl-D-galactosamine, generated the largest difference in fluorescence between infected and uninfected cells. The origin of the fluorescent signal emitted by infected cells was further investigated and attributed to the overexpression of glycoprotein on the surface of infected cells, as well as the presence of glycoprotein located in the proximity of intracellular parasites. Cryptosporidium parvum, an apicomplexan protozoan species, infects the intestine and sometimes the respiratory tract and bile duct of various mammalian hosts. In immunocompetent humans the infection causes self-limited diarrhea, but in immunocompromised individuals, primarily those with AIDS, the infection can become chronic and potentially cause lifethreatening syndromes (5). Infection with Cryptosporidium parasites occurs when food or water contaminated with oocysts is ingested, or possibly through the inhalation of oocysts. After entering the gastrointestinal tract of the host, four sporozoites released from the oocyst seek to invade the intestinal epithelial cells, where they multiply as meronts. Global gene expression profiles in cell monolayers infected with C. parvum have been studied using microarrays (3, 8, 20). These studies found that genes belonging to the cell proliferation, apoptosis, signal transduction, and transcription categories are overrepresented among significantly regulated genes (8, 20, 39). The overexpression of the osteoprotegerin gene in host cells after infection also was reported (3). Published microarray studies of C. parvum-infected cells are based on RNA extracted from cells in culture. The development of this parasite in culture is restricted in time, and the completion of the life cycle is only rarely observed (14, 32). High oocyst doses and long incubation times do not increase the proportion of infected cells (36) and may instead lead to the extensive perturbation of the monolayer caused primarily by apoptosis and mitosis (19, 21, 34, 38). Discerning which transcriptional changes occur directly in response to the infection, and which result from the perturbance of the monolayer (13, 36), is difficult. To eliminate the effect of partial infection and monolayer perturbation, we physically separated infected and uninfected cells to generate subpopulations that differed only with respect to the presence of intracellular parasites. We report on transcriptional and phenotypic changes identified by this approach, in particular the transcriptional upregulation and overexpression of cell surface glycoprotein in C. parvuminfected cells.