Expression of the highly polymorphic Cryptosporidium parvum Cpgp40/15 gene in genotype I and II isolates.
The enteric protozoan Cryptosporidium parvum infects intestinal epithelial cells in a wide range of hosts, causing severe gastrointestinal disease. The invasive sporozoite stage most likely attaches to and invades host cells through multiple host receptor/parasite ligand interactions. Preliminary evidence suggests that the glycoprotein products of the Cpgp40/15 gene, gp40 and gp15, are involved in these interactions. In addition, the Cpgp40/15 gene that encodes these glycopeptides is highly polymorphic in genotype I isolates, suggesting that the gene products may be subject to immune selection. In this study, we characterized the Cpgp40/15 gene in a genotype I isolate and compared expression of the Cpgp40/15 gene in isolates of both genotype. Cpgp40/15 is a single copy gene in both TU502 (genotype I) and GCH1 (genotype II) isolates. However, Northern blot analysis revealed the presence of two transcripts, 2.3 and 1.5 kb in size, in mRNA from GCH1 as well as TU502-infected Caco-2A cells. Accumulation of the two Cpgp40/15 mRNAs peaked 12-24 h post-infection. Using 3'RACE analysis, three polyadenylation sites were identified 371, 978 and 1002 bp downstream of the GCH1 Cpgp40/15 stop codon. Two of these polyadenylation sites were also used in TU502. The sequences of the GCH1 Cpgp40/15 3'untranslated regions (3'UTRs) were identical to genomic sequence and shared 96.7% homology with TU502 3'UTRs. Actinomycin D treatment of GCH1-infected Caco-2A cells followed by Northern blot analysis, revealed that the stability of the 1.5 kb message was considerably greater than that of the 2.3 kb transcript.
O'Connor
Roberta M
RM
Division of Geographic Medicine and Infectious Diseases, New England Medical Center, Tufts University School of Medicine, Box 041, 750 Washington Street, Boston, MA 02111, USA. roconnor@lifespan.org
Thorpe
Cheleste M
CM
Cevallos
Ana-Maria
AM
Ward
Honorine D
HD
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D018938:Caco-2 Cells; D002874:Chromosome Mapping; D016785:Cryptosporidium parvum / Q000235:genetics*; D003609:Dactinomycin / Q000494:pharmacology; D017125:Genes, Protozoan / Q000235:genetics*; D005838:Genotype; D006207:Half-Life; D006801:Humans; D008969:Molecular Sequence Data; D026723:Polyadenylation; D011110:Polymorphism, Genetic / Q000235:genetics*; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D020871:RNA Stability; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013997:Time Factors
C451425:Cpgp40-15 protein, Cryptosporidium parvum; D015800:Protozoan Proteins; D012333:RNA, Messenger; C106305:oocyst wall protein, Cryptosporidium; C069428:thrombospondin-related adhesive protein, protozoan; D003609:Dactinomycin