A role for host phosphoinositide 3-kinase and cytoskeletal remodeling during Cryptosporidium parvum infection

Cryptosporidium parvum preferentially infects epithelial cells lining the i ntestinal mucosa of mammalian hosts. Parasite development and propagation o ccurs within a unique intracellular but extracytoplasmic parasitophorous va cuole at the apical surface of infected cells. Parasite-induced host cell s ignaling events and subsequent cytoskeletal remodeling were investigated by using cultured bovine fallopian tube epithelial (BFTE) cells inoculated wi th C. parvum sporozoites. Indirect-immunofluorescence microscopy detected h ost tyrosine phosphorylation within 30 s of inoculation. At >30 min postino culation, actin aggregates were detected at the site of parasite attachment by fluorescein isothiocyanate-conjugated phalloidin staining as well as by indirect immunolabeling with monoclonal anti-actin. The actin-binding prot ein villin was also detected in focal aggregates at the site of attachment. Host cytoskeletal rearrangement persisted for the duration of the parasito phorous vacuole and contributed to the formation of long, branched microvil li clustered around the cryptosporidial vacuole. The phosphoinositide 3-kin ase inhibitor wortmannin significantly inhibited (P < 0.05) C parvum infect ion when BFTE cells were pretreated for 60 min at 37 degrees C prior to ino culation. Similarly, treatment of BFTE cells with the protein kinase inhibi tors genistein and staurosporine and the cytoskeletally acting compounds 1- (5-iodonaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazapine, cytochalasin D, and 2,3-butanedione monoxime significantly inhibited (P < 0.05) in vitro in fection at 24 h postinoculation. These findings demonstrate a prominent rol e for phosphoinositide 3-kinase activity during the early C. parvum infecti on process and suggest that manipulation of host signaling pathways results in actin rearrangement at the site of sporozoite attachment.