Marie Labbé

Identification of the nucleic acid binding domain of the rotavirus VP2 protein

The bovine rotavirus VP2 protein is the major component of the core and forms the most internal layer surrounding the dsRNA genome. We have constructed recombinant baculoviruses expressing truncated VP2 proteins. The nucleic acid binding activity of these truncated proteins was tested by North-Western blotting experiments with single-stranded and double-stranded probes. The nucleic acid binding domain in VP2 was localized between amino acids 1 to 132. Recombinant proteins bound single-stranded and double-stranded nucleic acids, but showed less affinity for double-stranded RNA and DNA. Interactions of VP2 with the genome were investigated in viral single-shelled particles by u.v.-cross-linking. In these experiments, only VP2 protein bound the genomic RNA in purified single-shelled particles.

An immunodominant cytotoxic T cell epitope on the VP7 rotavirus protein overlaps the H2 signal peptide

C57BL/6 (H-2b) mice were primed with the bovine RF strain of rotavirus to study the induction of CD8+ cytotoxic T lymphocytes (CTLs). These rotavirus-specific CTLs were detected only after in vitro restimulation with the virus. Using a recombinant vaccinia virus we identified the RF VP7 protein as a major target of these CTLs. The response against this protein was obtained also after in vitro restimulation with simian SA11 and human WA strains of rotavirus. Using published Db and Kb allele-specific motifs to predict possible CTL epitopes in the RF VP7 protein, we synthesized and tested 18 predicted peptides of VP7. Only one peptide was able to sensitize target cells at a concentration below 5 x 10(-7) M. This CTL epitope was also induced by immunization with the RF VP7 expressed with a baculovirus vector, and was shown to be immunodominant by its capacity to inhibit, in an unlabelled target assay, the bulk response against cells infected with recombinant vaccinia virus expressing VP7. This CTL epitope overlaps the H2 signal peptide of the protein.

Differential localisation of an Eimeria tenella aspartyl proteinase during the infection process

Aspartyl proteinases are essential for the survival of many pathogens. A single copy gene in species of Eimeria encodes an aspartyl proteinase, which we propose should be called eimepsin to conform to the commonly used names of this family of proteinases. An epitope map, constructed using BIAcore technology, confirmed the specificity of 14 mAbs for eimepsin and defined four antigenic domains, which were conserved between native and recombinant forms of eimepsin. In resting sporozoites, mAb defining antigenic domains I and II stained the refractile body organelles, whereas those defining antigenic domains III and IV stained cytoplasmic granules. During host cell invasion, the staining patterns of mAb defining antigenic domains I, III and IV changed dramatically with the apical tips of invading sporozoites becoming strongly stained. In contrast, mAb defining antigenic domain II continued to stain only the refractile bodies. During early schizogony, mAb to all four domains stained the single fused refractile body, but when schizonts matured, mAb to antigenic domains I, III and IV stained the apical tip of merozoites whereas those to antigenic domain II continued to follow the developmental redistribution of the refractile body. Irrespective of localisation, mAb to three antigenic domains recognised a polypeptide of 49 kDa, which from N-terminal sequencing corresponds to a mature form of eimepsin. Staining with fluorescent pepstatin localised a mature, active form of eimepsin to the refractile bodies of the sporozoite, schizont and first generation merozoite. It remains to be determined whether eimepsin has a catalytic function within the refractile body or whether the activated enzyme is stored in the refractile body so that it can be rapidly redistributed to the apical tip during parasite invasion.

Eimeria tenella: cloning and characterization of cDNA encoding a S3a ribosomal protein

A lambda Zap II cDNA library was constructed from Eimeria tenella first- generation schizonts mRNA and screened with a mouse serum raised against this parasitic stage. This serum identified a clone encoding a S3a ribosomal protein (EtS3a). The 858-bp cDNA fragment, containing the entire parasitic gene encoded a highly basic protein of 264 amino acids (aa) with a molecular weight of 29.780kDa. Based upon amino acid sequence comparison, EtS3a is highly homologous to v-fos transformation effector (encoded by the fte-1 gene) and cyc-07 (a plant homologue of fte-1) and similar to the yeast MFT1 (encoded by the mitochondrial fusion targeting gene). The expressions of mammalian fte-1, plant cyc-07 and yeast MFT1 have all been shown to be cell-cycle-regulated and involved in protein synthesis at the level of the ribosome. Since EtS3a expression is also developmentally regulated, we suggest that this gene product is a functional homologue of fte-1, cyc-07 and MFT1 and an important molecule regulating the development of Eimeria tenella.

Gene discovery in Eimeria tenella by immunoscreening cDNA expression libraries of sporozoites and schizonts with chicken intestinal antibodies

Specific antibodies were produced ex vivo from intestinal culture of Eimeria tenella infected chickens. The specificity of these intestinal antibodies was tested against different parasite stages. These antibodies were used to immunoscreen first generation schizont and sporozoite cDNA libraries permitting the identification of new E. tenella antigens. We obtained a total of 119 cDNA clones which were subjected to sequence analysis. The sequences coding for the proteins inducing local immune responses were compared with nucleotide or protein databases and with expressed sequence tags (ESTs) databases. We identified new Eimeria genes coding for heat shock proteins, a ribosomal protein, a pyruvate kinase and a pyridoxine kinase. Specific features of other sequences are discussed.

PURIFICATION OF FIRST-GENERATION EIMERIA TENELLA SCHIZONTS

A rapid and simple method for purifying first-generation Eimeria tenella schizonts was developed with infected chicken cecal tissue. The schizonts were harvested from the tissue by treatment with a mixture of 3 enzymes: hyaluronidase, dispase, and collagenase. Subsequent purification of the parasites by filtration through gauze and centrifugation resulted in a clean final preparation compared with the starting material. Microscopical and biochemical examinations, performed to assess the quality of the schizont purification, showed a high degree of purity. This procedure may be used to obtain clean schizonts minimally contaminated with host-cell debris in order to study this parasitic stage.