title: Role of CpSUB1, a subtilisin-like protease, in Cryptosporidium parvum infection in vitro.
abstract: The apicomplexan parasite Cryptosporidium is a significant cause of diarrheal disease worldwide. Previously, we reported that a Cryptosporidium parvum subtilisin-like serine protease activity with furin-type specificity cleaves gp40/15, a glycoprotein that is proteolytically processed into gp40 and gp15, which are implicated in mediating infection of host cells. Neither the enzyme(s) responsible for the protease activity in C. parvum lysates nor those that process gp40/15 are known. There are no furin or other proprotein convertase genes in the C. parvum genome. However, a gene encoding CpSUB1, a subtilisin-like serine protease, is present. In this study, we cloned the CpSUB1 genomic sequence and expressed and purified the recombinant prodomain. Reverse transcriptase PCR analysis of RNA from C. parvum-infected HCT-8 cells revealed that CpSUB1 is expressed throughout infection in vitro. In immunoblots, antiserum to the recombinant CpSUB1 prodomain revealed two major bands, of approximately 64 kDa and approximately 48 kDa, for C. parvum lysates and proteins "shed" during excystation. In immunofluorescence assays, the antiserum reacted with the apical region of sporozoites and merozoites. The recombinant prodomain inhibited protease activity and processing of recombinant gp40/15 by C. parvum lysates but not by furin. Since prodomains are often selective inhibitors of their cognate enzymes, these results suggest that CpSUB1 may be a likely candidate for the protease activity in C. parvum and for processing of gp40/15. Importantly, the recombinant prodomain inhibited C. parvum infection of HCT-8 cells. These studies indicate that CpSUB1 plays a significant role in infection of host cells by the parasite and suggest that this enzyme may serve as a target for intervention.
authors.affiliation: Division of Geographic Medicine and Infectious Diseases, Tufts Medical Center, Boston, MA 02111, USA.
authors.forename: Jane W
authors.initials: JW
authors.lastname: Wanyiri
authors.forename: Patsharaporn
authors.initials: P
authors.lastname: Techasintana
authors.forename: Roberta M
authors.initials: RM
authors.lastname: O'Connor
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Blackman
authors.forename: Kami
authors.initials: K
authors.lastname: Kim
authors.forename: Honorine D
authors.initials: HD
authors.lastname: Ward
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D003457:Cryptosporidiosis / Q000469:parasitology*; D016785:Cryptosporidium parvum / Q000201:enzymology / Q000235:genetics / Q000254:growth & development / Q000502:physiology*; D006790:Host-Parasite Interactions; D006801:Humans; D008970:Molecular Weight; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D020860:Subtilisin / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D015800:Protozoan Proteins; D020860:Subtilisin