title: Cryptosporidium parvum infects human cholangiocytes via sphingolipid-enriched membrane microdomains.
abstract: Cryptosporidium parvum attaches to intestinal and biliary epithelial cells via specific molecules on host-cell surface membranes including Gal/GalNAc-associated glycoproteins. Subsequent cellular entry of this parasite depends on host-cell membrane alterations to form a parasitophorous vacuole via activation of phosphatidylinositol 3-kinase (PI-3K)/Cdc42-associated actin remodelling. How C. parvum hijacks these host-cell processes to facilitate its infection of target epithelia is unclear. Using specific probes to known components of sphingolipid-enriched membrane microdomains (SEMs), we detected aggregation of host-cell SEM components at infection sites during C. parvum infection of cultured human biliary epithelial cells (i.e. cholangiocytes). Activation and membrane translocation of acid-sphingomyelinase (ASM), an enzyme involved in SEM membrane aggregation, were also observed in infected cells. Pharmacological disruption of SEMs and knockdown of ASM via a specific small interfering RNA (siRNA) significantly decreased C. parvum attachment (by approximately 84%) and cellular invasion (by approximately 88%). Importantly, knockdown of ASM and disruption of SEMs significantly blocked C. parvum-induced accumulation of Gal/GalNAc-associated glycoproteins at infection sites by approximately 90%. Disruption of SEMs and knockdown of ASM also significantly blocked C. parvum-induced activation of host-cell PI-3K and subsequent accumulation of Cdc42 and actin by up to 75%. Our results suggest an important role of SEMs for C. parvum attachment to and entry of host cells, likely via clustering of membrane-binding molecules and facilitating of C. parvum-induced actin remodelling at infection sites through activation of the PI-3K/Cdc42 signalling pathway.
authors.affiliation: Center for Basic Research in Digestive Diseases, Division of Gastroenterology and Hepatology, Mayo Clinic College of Medicine, Rochester, MN 55905, USA.
authors.forename: Jeremy B
authors.initials: JB
authors.lastname: Nelson
authors.forename: Steven P
authors.initials: SP
authors.lastname: O'Hara
authors.forename: Aaron J
authors.initials: AJ
authors.lastname: Small
authors.forename: Pamela S
authors.initials: PS
authors.lastname: Tietz
authors.forename: Amit K
authors.initials: AK
authors.lastname: Choudhury
authors.forename: Richard E
authors.initials: RE
authors.lastname: Pagano
authors.forename: Xian-Ming
authors.initials: XM
authors.lastname: Chen
authors.forename: Nicholas F
authors.initials: NF
authors.lastname: LaRusso
mesh_terms: D000199:Actins / Q000378:metabolism; D000818:Animals; D001653:Bile Ducts, Intrahepatic / Q000166:cytology / Q000469:parasitology*; D002460:Cell Line; D003457:Cryptosporidiosis / Q000378:metabolism*; D016785:Cryptosporidium parvum / Q000472:pathogenicity*; D004847:Epithelial Cells / Q000469:parasitology*; D006801:Humans; D053592:Interleukin-1 Receptor-Associated Kinases / Q000378:metabolism; D021962:Membrane Microdomains / Q000378:metabolism*; D013107:Sphingolipids / Q000737:chemistry / Q000378:metabolism*; D020764:cdc42 GTP-Binding Protein / Q000378:metabolism
chemical_list: D000199:Actins; D013107:Sphingolipids; D053592:Interleukin-1 Receptor-Associated Kinases; D020764:cdc42 GTP-Binding Protein