Analysis of Sequence Diversity at the Highly Polymorphic Cpgp40 / 15 Locus among Cryptosporidium Isolates from Human Immunodeficiency Virus-Infected Children in South Africa

Abstract

Cryptosporidium sp. is a significant cause of diarrheal disease, particularly in human immunodeficiency virus (HIV)-infected patients in developing countries. We recently cloned and sequenced several alleles of the highly polymorphic single-copy Cryptosporidium parvum gene Cpgp40/15. This gene encodes a precursor protein that is proteolytically cleaved to yield mature cell surface glycoproteins gp40 and gp15, which are implicated in zoite attachment to and invasion of enterocytes. The most-striking feature of the Cpgp40/15 alleles and proteins is their unprecedented degree of sequence polymorphism, which is far greater than that observed for any other gene or protein studied in C. parvum to date. In this study we analyzed nucleic acid and amino acid sequence polymorphism at the Cpgp40/15 locus of 20 C. parvum isolates from HIV-infected South African children. Fifteen isolates exhibited one of four previously identified genotype I alleles at the Cpgp40/15 locus (Ia, Ib, Ic, and Id), while five displayed a novel set of polymorphisms that defined a new Cpgp40/15 genotype I allele, designated genotype Ie. Surprisingly, only 15 of these isolates exhibited concordant type I alleles at the thrombospondin-related adhesive protein of Cryptosporidium and Cryptosporidium oocyst wall protein loci, while five isolates (all of which displayed Cpgp40/15 genotype Ic alleles) displayed genotype II alleles at these loci. Furthermore, the last five isolates also manifested chimeric genotype Ic/Ib or Ic/II alleles at the Cpgp40/15 locus, raising the possibility of sexual recombination within and between prototypal parasite genotypes. Lastly, children infected with isolates having genotype Ic alleles were significantly older than those infected with isolates displaying other genotype I alleles.

Whole genome amplification (WGA) for archiving and genotyping of clinical isolates ofCryptosporidiumspecies

“…Several subtyping techniques have been applied to Cryptosporidium species using different markers: glycoprotein GP60 (Strong et al 2000 ;Leav et al 2002), double-stranded RNA element (Leoni et al 2003) and mini-and microsatellite repeats (Cacciò et al 2000 ;Mallon et al 2003). Ideally, each new isolate should be tested using a panel of markers.…”

Symptomatic and Asymptomatic Cryptosporidium Infections in Children in a Semi-Urban Slum Community in Southern India

“…Initially, three sequentially collected surveillance samples from each child were pooled and resulted in 343 pools derived from 1,036 samples, DNA was extracted from the pool and analyzed by PCR. From all positive pools, DNA was re-extracted from individual stool samples, and the infecting species and subgenotype were identified by PCR-RFLP at the SSU rRNA locus 20 and the Cpgp40/15 locus, 22 respectively, by using previously described protocols. Diarrheal stool samples were also screened for parasite ova and cysts by microscopy, for bacterial pathogens by culture, and for rotavirus by enzyme-linked immunosorbent assay (Rota IDEIA; Dako, Ely, United Kingdom).…”

Human CD8+ T Cells Clear Cryptosporidium parvum from Infected Intestinal Epithelial Cells

“…The gp15 was produced at Tufts University as previously described. 15,19 We used C. hominis gp15, which is highly homologous to C. parvum gp15, 20,21 and has been shown to elicit stronger IFN-γ responses in sensitized persons. 15 Cells were incubated (37°C, 5% CO 2 ) for 6 days.…”