Characterization and immunolocalization of a Cryptosporidium protein containing repeated amino acid motifs

Abstract

The oocyst wall is one of the components that permits cryptosporidia both to survive in the environment and to retain infectivity. With the aim of identifying Cryptosporidium proteins specifically expressed at the oocyst stage, we screened Agtll genomic libraries of Cryptosporidium parvum with both an oocyst antiserum and a * Corresponding author.We thought that such genes would be important both in understanding how the parasites survive and retain infectivity for several months in the external environment and in searching for the antigens that may be the targets of the immune response. To generate Xgtll expression libraries of cryptosporidia, we used either DNA extracted from purified oocysts or DNA derived from the gut cells of the brush border of a Cryptosporidium parvum-infected calf. For this purpose, we developed a method for collecting the infected cells of the superficial layer of the mucosa, thus increasing the parasite DNA/intestinal cell DNA ratio. On the basis of the reactivity with a Cryptosporidium oocyst antiserum, we selected from the library insert cpRL3, which was generated with the DNA extracted from infected gut cells. This insert encoded a partial sequence of 786 amino acids. The analysis of the deduced amino acid sequence suggested that the Cryptosporidium protein may play an important functional and/or structural role in the parasite. Using cpRL3 as a hybridization probe, we isolated from a library generated with shared DNA of Cryptospondium oocysts an insert of 4,420 bp. This insert encompassed the entire sequence of cpRL3 and contained an open reading frame of 1,252 amino acids. The deduced amino acid sequence was characterized by a high content of cysteine and by the presence of two distinct repeated amino acid motifs. We developed sera and monoclonal antibodies against a recombinant polypeptide encompassing the first 786 amino acids to study the expression and localization of the protein in C. parvum. MATERIALS AND METHODS Developmentof a Agtll library with DNA extracted from infected gut mucosa. To develop a C. parvum genomic 2347 on July 16, 2020 by guest http://iai.asm.org/ Downloaded from 2348 RANUCCI ET AL. on July 16, 2020 by guest http://iai.asm.org/ Downloaded from

Calcium-Mediated Biophysical Binding of Cryptosporidium parvum Oocysts to Surfaces Is Sensitive to Oocyst Age

“…4b are consistent with an ionic bridging binding process that arises due to EDL events (10,11,14,45,46); however, the magnitude of the force indicates that various surface characteristics of the oocysts may contribute to these interactions (11,12). The C. parvum wall structure has been reported to be multilayered, with the outer layer of the wall covered in acidic glycoproteins (6,54,55); the outer-wall structure exhibits an isoelectric point of ϳ2.5, thus exhibiting a negative charge at the experimental pH of 6.8. In addition, this outer wall possesses a glucose-rich glycocalyx, which likely plays a role in nonspecific physiochemical binding.…”

Detection of Cryptosporidium parvum in raw milk by PCR and oligonucleotide probe hybridization

“…Ranucci et al (33) cloned a DNA fragment encoding an oocyst protein (COWP-190) of C. parvum. The first 2,359 bp of their clone was identical to the DNA sequence reported by Lally et al (22).…”

Evaluation of PCR, nested PCR, and fluorescent antibodies for detection of Giardia and Cryptosporidium species in wastewater

“…Detection of Cryptosporidium organisms by PCR required a nested step since the primers published by Ranucci et al (20) for the 753-bp amplicon resulted in nonspecific amplification, as evidenced by the numerous nontarget bands in Fig. 1a, and did not allow visualization of the target 753-bp band.…”

“…Concurrently, the sewage samples were analyzed for Giardia and Cryptosporidium organisms by PCR. Primers reported by Mahbubani et al (12) were used for Giardia detection while a nested PCR based on primers reported by Ranucci et al (20) was developed for Cryptosporidium detection.…”