Characterization of subtilase protease in Cryptosporidium parvum and C. hominis.
Cryptosporidium spp., enteropathogens of humans and other animals, are members of the Apicomplexa. In parasites belonging to this phylum, proteases have been shown to play a key role in the invasion of host cells, organelle biogenesis, and intracellular survival. The subtilases constitute a family of serine proteases present in prokaryotes, eukaryotes, and viruses. The C. parvum subtilase gene, CpSUB1, encodes a transcript of 3972 base pairs (bp) and 1324 amino acids. Using homologous polymerase chain reaction primers, a similar gene, ChSUB1, which has 98% (4007 bp/4050 bp) identity to CpSUB1, was found in C. hominis. The alignment of the CpSUB1 and ChSUB1 nucleotide sequences identified primarily silent substitutions, consistent with the absence of diversifying selection. The catalytic domain of CpSUB1 is very similar to that of other Apicomplexa (> 38% amino acid identity and >57% similarity) and to the bacterial subtilisin BPN from B. subtilis (36 and 47%). Transcriptional upregulation during merozoite development was observed in cell culture, and a predicted 76-bp intron located near the 3' end of the open reading frame was confirmed experimentally. Cryptosporidium parvum infection in cell culture was significantly inhibited by subtilisin inhibitor III and other serine protease inhibitors, emphasizing the importance of the parasite's subtilase for intracellular development and the enzyme's potential as a drug target.
Feng
Xiaochuan
X
Division of Infectious Diseases, Tufts Cummings School of Veterinary Medicine, North Grafton, Massachusetts 01536, USA.
Akiyoshi
Donna E
DE
Widmer
Giovanni
G
Tzipori
Saul
S
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002460:Cell Line; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D003458:Cryptosporidium / Q000187:drug effects / Q000201:enzymology* / Q000254:growth & development; D016785:Cryptosporidium parvum / Q000187:drug effects / Q000201:enzymology* / Q000254:growth & development; D005856:Germ-Free Life; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016415:Sequence Alignment; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000235:genetics; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D013552:Swine
D015842:Serine Proteinase Inhibitors; D012697:Serine Endopeptidases