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Eukaryotic Cell, April 2009, p. 470-477, Vol. 8, No. 4
1535-9778/09/$08.00+0     doi:10.1128/EC.00306-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Role of CpSUB1, a Subtilisin-Like Protease, in Cryptosporidium parvum Infection In Vitro{triangledown}

Jane W. Wanyiri,1 Patsharaporn Techasintana,1 Roberta M. O'Connor,1 Michael J. Blackman,2 Kami Kim,3 and Honorine D. Ward1*

Division of Geographic Medicine and Infectious Diseases, Tufts Medical Center, 800 Washington St., Boston, MA 02111,1 Division of Parasitology, The National Institute for Medical Research, London, United Kingdom,2 Departments of Medicine and Immunology and Microbiology, Albert Einstein College of Medicine, New York, New York3

Received 11 September 2008/ Accepted 21 November 2008

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 ~64 kDa and ~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.

* Corresponding author. Mailing address: Division of Geographic Medicine and Infectious Diseases, Tufts Medical Center, 800 Washington St., Boston, MA 02111. Phone: (617) 636-7022. Fax: (617) 636-5292. E-mail: Hward{at}tuftsmedicalcenter.org

{triangledown} Published ahead of print on 23 January 2009.

Eukaryotic Cell, April 2009, p. 470-477, Vol. 8, No. 4
1535-9778/09/$08.00+0     doi:10.1128/EC.00306-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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