Bile Acids Enhance Invasiveness of Cryptosporidium spp. into Cultured Cells

Abstract

Bile salts such as sodium taurocholate (NaTC) are routinely used to induce the excystation of Cryptosporidium oocysts. Here we show that NaTC significantly enhanced the invasion of several cultured cell lines by freshly excysted Cryptosporidium parvum and Cryptosporidium hominis sporozoites. A variety of purified bile salts or total bile from bovine also enhanced the invasion of cultured cells by C. parvum. Further studies demonstrated that NaTC increased protein secretion and gliding motility of sporozoites, the key processes for successful invasion. These observations may lead to improved Cryptosporidium infectivity of cultured cells and help future studies on the host-parasite interaction.As members of the phylum Apicomplexa, the enteric protozoa Cryptosporidium species share a common apical secreting apparatus that mediates locomotion during cellular invasion (5, 21). Cryptosporidium hominis and Cryptosporidium parvum, whose genomes were sequenced recently (1, 28), are the species most frequently linked with human cryptosporidiosis (21,27). Infection begins with the oral uptake of Cryptosporidium oocysts, the environmentally resistant form, which then pass through the acidic stomach before entering the small intestine, where they presumably excyst. Although the molecular and biochemical mechanisms involved in excystation are poorly understood, it is believed that host environmental factors, such as temperature, pH, proteases, bile salts, and possibly other unknown factors, trigger the excystation of ingested oocysts (19). The newly excysted sporozoites secrete adhesive molecules and other signaling proteins which have been demonstrated to play a key role in the initial attachment and cellular invasion (4, 6).The in vitro cell culture system, while limited to the asexual phase, has been a useful tool for investigating early parasite attachment and invasion and has been applied to measure Cryptosporidium infectivity and for screening of compounds for inhibitory activity (3,17). Several cell lines, including human ileocecal adenocarcinoma (HCT-8), human colonic adenocarcinoma (Caco-2), and Madin-Darby bovine kidney (MDBK) cell lines, are widely used for Cryptosporidium in vitro studies (12,22). To initiate in vitro infection, purified Cryptosporidium oocysts are often treated with sodium hypochlorite either alone or followed by sodium taurocholate (NaTC)-trypsin treatment before infecting cell monolayers. Several publications have dealt with assessment of the conditions and materials for optimal oocyst excystation and tissue culture infection by the excysted sporozoites (15,16,23,24). Upton et al. (24) investigated the optimization of infection of oocysts treated with bleach without prior excystation. Gold et al. showed that the presence of NaTC in the culture medium enhanced infection when oocysts were directly added to cell culture monolayers (11). However, the nature of this enhancement was not fully investigated, and it was assumed that the NaTC facilitated the excystation of oocysts in the culture medium (...

The Terminal Sialic Acid of Glycoconjugates on the Surface of Intestinal Epithelial Cells Activates Excystation of Cryptosporidium parvum

“…Excystation in the presence of both bile salt and Caco2 cells, however, was significantly better than in samples containing cells only. Bile salts may have a separate major role in parasite infection by activating sporozoite gliding required for host cell invasion (8).…”

“…Both raised temperature and acidic conditions have been demonstrated to increase the permeability of the oocyst wall to small molecules (20). It has been established that bile salts increase the rate of excystation, and a study reported that, in addition, sodium deoxycholate improved the invasiveness of sporozoites for epithelial cells (8). Increased parasite protease activity during excystation has been described, and sporozoite release was hindered by protease inhibitors (9).…”

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

“…Subconfluent HCT-8 monolayers seeded in T75 flasks were infected with C. parvum oocysts at a 1:4 oocyst/host cell ratio and incubated for 24 h. This time point was chosen because at later time points monolayer perturbance increases. Taurocholic acid at a concentration of 0.05% was added with the oocysts to promote infection (11). After incubation, cell monolayers were recovered by treatment with Accutase (Innovative Cell Technologies, Inc.) and immunofluorescently labeled as described previously (31), except that polyclonal rabbit antibody specific for C. parvum sporozoites and oocysts was used as the primary antibody and the host cell membrane was not permeabilized.…”

Human Primary Intestinal Epithelial Cells as an Improved In Vitro Model for Cryptosporidium parvum Infection

“…However, progress in research on these organisms has been hampered by the limitations of current experimental models (6). Because animal models are suboptimal for some human infections, human colonic cell lines have been used as an alternative to study intestinal pathogens (7,8). The utility of cell lines is limited by the fact they are not derived from the small intestine and are transformed.…”