Expression of the highly polymorphic Cryptosporidium parvum Cpgp40/15 gene in genotype I and II isolates

Roberta M. O'Connor, Cheleste M. Thorpe, Ana Maria Cevallos, Honorine D. Ward

Research output: Contribution to journalArticlepeer-review

17 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)203-215
Number of pages13
JournalMolecular and Biochemical Parasitology
Volume119
Issue number2
DOIs
StatePublished - 2002
Externally publishedYes

Bibliographical note

Funding Information:
We wish to thank Dr Saul Tzipori and Dr Donna Akiyoshi for the GCH1 and TU502 isolates, the TU502 gDNA and the GCH1 gDNA library, Smitha Jaison for excellent technical assistance and Anne Kane for helpful comments and careful review of the manuscript. This work was supported by Grant AI46299 from the NIAID, NIH; Grant 2000-02247 from the USDA; and Grant P30 DK34928 from the NIDDK, NIH to the GRASP Digestive Diseases Center at New England Medical Center. R.M. O'Connor was supported by training Grant AI07389 from the NIAID, NIH.

Keywords

  • 3′UTRs
  • Alternate polyadenylation
  • Cpgp40/15
  • Cryptosporidium
  • Polymorphisms
  • mRNA stability

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