Cryptosporidium parvum infects human cholangiocytes via sphingolipid-enriched membrane microdomains.
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.
Nelson
Jeremy B
JB
Center for Basic Research in Digestive Diseases, Division of Gastroenterology and Hepatology, Mayo Clinic College of Medicine, Rochester, MN 55905, USA.
O'Hara
Steven P
SP
Small
Aaron J
AJ
Tietz
Pamela S
PS
Choudhury
Amit K
AK
Pagano
Richard E
RE
Chen
Xian-Ming
XM
LaRusso
Nicholas F
NF
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
D000199:Actins; D013107:Sphingolipids; D053592:Interleukin-1 Receptor-Associated Kinases; D020764:cdc42 GTP-Binding Protein