Proteomic Array Analysis of an Epithelial Cell Model Infected with Cryptosporidium

摘要

Cryptosporidiosis is a human diarrheal disease caused by the etiological agent Cryptosporidium. This parasite abounds in the environment in resistant oocyst form and can cause life-threatening consequences in immunosuppressed individuals. By evaluating the fundamental mechanisms of pathogenesis in the host, it is helpful to deduce proteins involved in virulence. Parasite-induced host cell signaling events and subsequent cytoskeletal remodeling were investigated by using cultured human ileocecal carcinoma epithelial (HCT-8) cells inoculated with C. parvum sporozoites. Although many efforts have been made to characterize gene expression changes, these changes may not necessarily reflect changes in protein expression within an infected cell. New applications in proteomic arrays have the added benefit of studying changes in protein expression and functionality. In this study, we use a protein array platform to observe changes in protein expression and function when host cells are infected with C. parvum sporozoites. Previous studies have shown that the invasion of epithelial cells involves host-signaling whereby host cell actin reorganization allows for the enveloping of the parasite. A major signaling pathway implicated in actin polymerization involves the activation and deactivation of proteins containing SH2 domains, such as CRKL, PIK$3ABL, and the Src protein tyrosine kinase (PTK) family. Our studies show that a phospho-proteomic array of HCT-8 cells inoculated with the pathogen have a 0.87-fold increase in the phosphorylated form of c-Abl. Moreover, an SH2 proteomic array overlayed with protein lysates from uninfected and infected cells revealed that infected protein lysates had decreased binding (66%) to the SH2 domain of CRKL and increased binding (45%) to the SH2 domain of PIK3. These findings demonstrate a prominent role for proteins that may play a role in formation and stabilization of microvillar protrusions which envelope the pathogen.