Molecular cloning and immunoelectron microscopy have been used to clone the full-length gene encoding Cryptosporidium parvum oocyst wall protein (COWP) and to analyse at the ultrastructural level the expression and localization of COWP during development in the gut. COWP is 1622 amino acids long, has a typical leader peptide and consists of 2 amino acidic domains each containing distinct repeated elements possibly originating from a common ancestral precursor. Electron microscopy localized COWP in a large cytoplasmic inclusion and in the wall-forming bodies of early and late macrogametes, respectively. Ultrastructural analysis of double-walled sporulating and mature oocysts indicated that COWP is selectively localized in the inner layer of the oocyst wall. This study provides the first localization at the ultrastructural level of a cloned coccidian oocyst wall protein.

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

By screening an expression library of the yeast form of Candida albicans with a serum directed against whole fungal cells, a cDNA (2,325 bp) encoding a stress protein of C. albicans was cloned and sequenced. The cloned sequence (CaRLV130) identified a single open reading frame with a length of 1,968 bp coding for a protein containing 656 amino acid residues (70 kDa). The deduced amino acid sequence was 84% similar to the sequence of the Saccharomyces cerevisiae SSA1 gene, which encodes one member of the 70-kDa heat shock protein (Hsp70) family. The relevant gene (C. albicans HSP70 gene [CaHSP70]) was localized on the highest-M r (R1; approximately 3.8 Mb) chromosome of C. albicans as determined by pulse-field electrophoresis. CaHSP70 was expressed after heat shock, as demonstrated by Northern (RNA) blotting and reverse transcriptase-PCR with specific pairs of oligonucleotide sequences and gene probes. A recombinant protein was obtained in Escherichia coli after cloning of the full coding sequence into the BamHI site of the pDS56/RBSII6xhisE ؊ plasmid and purification by nickel chelate affinity chromatography. The recombinant protein (6xhis-CaHsp70) was efficiently recognized in immunoblots by a monoclonal antibody directed against a common epitope of eukaryotic Hsp70 proteins, as well as by sera from normal human subjects. Moreover, immune mouse sera against the purified recombinant protein recognized native, heat-inducible constituents with sizes of around 70 kDa in whole-cell protein extracts of C. albicans. Overall, our data demonstrate that CaHSP70 encodes one member of a family of proteins (Hsp70) which usually represent highly conserved immunodominant antigens of infectious agents.

26:524-529, 1988). In order to define the virulence potential of these strains, we characterized the replication properties of their high-molecular-weight plasmids and studied the genetic locations and organization of the aerobactin (aer) and hemolysin (hly) determinants encoded by 23 NLF 04 E. coli strains. Southern blot hybridizations, mobilization assays of nonconjugative plasmids, and incompatibility-exclusion experiments conducted with a conjugative incompatibility group FT (IncFI) plasmid showed that (i) 20 out of the 23 strains examined harbor a 160-to 180-kb IncFI plasmid that shares homology with the basic replicons RepFIA, RepFIB, and (except for the plasmid of one strain) RepFIC, and 22 strains also contain a 40to 140-kb IncFIl plasmid sharing homology with the RepFlIA replicon; (ii) the IncFI plasmid is nonconjugative and carries antibiotic resistance genes; (iii) the aer system is located on the IncFI plasmids and/or the chromosomes in the three strains not harboring IncFI, and it is found in an inverted orientation; (iv) the hly determinants are located on the chromosome, and their genetic organization is well conserved and on August 2, 2020 by guest http://iai.asm.org/ Downloaded from

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