Cryptosporidium parvum invasion of biliary epithelia requires host cell tyrosine phosphorylation of cortactin via c-Src.
OBJECTIVE
Cryptosporidium parvum invasion of epithelia requires polymerization of host cell actin at the attachment site. We analyzed the role of host cell c-Src, a cytoskeleton-associated protein tyrosine kinase, in C. parvum invasion of biliary epithelia.


METHODS
In vitro models of biliary cryptosporidiosis using a human biliary epithelial cell line were used to assay the role of c-Src signaling pathway in C. parvum invasion.


RESULTS
c-Src and cortactin, an actin-binding protein and a substrate for c-Src, were recruited to the parasite-host cell interface during C. parvum invasion. Tyrosine phosphorylation of cortactin in infected cells was also detected. Inhibition of host cell c-Src significantly blocked C. parvum -induced accumulation and tyrosine phosphorylation of cortactin and actin polymerization at the attachment sites, thereby inhibiting C. parvum invasion of biliary epithelial cells. A triple mutation of tyrosine of cortactin in the epithelia also diminished C. parvum invasion. In addition, proteins originating from the parasite were detected within infected cells at the parasite-host cell interface. Antiserum against C. parvum membrane proteins blocked accumulation of c-Src and cortactin and significantly decreased C. parvum invasion. No accumulation of the endocytosis-related proteins, dynamin 2 and clathrin, was found at the parasite-host cell interface; also, inhibition of dynamin 2 did not block C. parvum invasion.


CONCLUSIONS
C. parvum invasion of biliary epithelial cells requires host cell tyrosine phosphorylation of cortactin by a c-Src-mediated signaling pathway to induce actin polymerization at the attachment site, a process associated with microbial secretion but independent of host cell endocytosis.
Chen
Xian-Ming
XM
Center for Basic Research in Digestive Diseases, Division of Gastroenterology and Hepatology, Mayo Medical School, Clinic and Foundation, Rochester, Minnesota 55905, USA.
Huang
Bing Q
BQ
Splinter
Patrick L
PL
Cao
Hong
H
Zhu
Guan
G
McNiven
Mark A
MA
LaRusso
Nicholas F
NF
D000199:Actins / Q000378:metabolism; D000818:Animals; D000913:Antibodies, Protozoan / Q000494:pharmacology; D001652:Bile Ducts / Q000166:cytology / Q000469:parasitology; D000081247:CSK Tyrosine-Protein Kinase; D002461:Cell Line, Transformed; D051356:Cortactin; D003457:Cryptosporidiosis / Q000378:metabolism*; D016785:Cryptosporidium parvum / Q000276:immunology / Q000472:pathogenicity*; D003599:Cytoskeleton / Q000378:metabolism; D004705:Endocytosis; D004847:Epithelial Cells / Q000166:cytology / Q000201:enzymology / Q000469:parasitology*; D006790:Host-Parasite Interactions; D006801:Humans; D008840:Microfilament Proteins / Q000378:metabolism*; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011817:Rabbits; D014443:Tyrosine / Q000378:metabolism; D014774:Virulence; D019061:src-Family Kinases
D000199:Actins; D000913:Antibodies, Protozoan; C495868:CTTN protein, human; D051356:Cortactin; D008840:Microfilament Proteins; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D000081247:CSK Tyrosine-Protein Kinase; D019061:src-Family Kinases; C000631346:CSK protein, human