ANALYSIS OF INTRACELLULAR ADAPTATION MECHANISMS OF PROTOZOAN PARASITES AND HOST ELIMINATION

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This project was aiming at clarifying mechanisms of parasite adaptation to host cell environment which will lead us to establish control methods of intracellular parasite infections. We cloned a gene encoding small subunit of ribonucleotide reductase (R2) which is involved denovo synthesis of nucleotides. To clone the gene from Theileria sergenti, oligonucleotide primers for polymerase chain reaction (PCR) were designed based on the corresponding gene sequence of T.annulata. As a reverse transcription-PCR product contained a partial fragment of the gene, 5'-3' RACE (rapid amplification of cDNA ends) were carried out to obtain a full length mRNA sequence. A genomic clone containing R2 gene was also obtained by screening of T.sergenti genomic DNA library with cDNA probe. Sequence analysis of the genomic DNA clone revealed that the R2 gene contains at least 2 introns. Predicted amino acid sequence of R2 C-terminal portion showed significant difference from that of host R2, indicating that inhibitory peptides specific for the parasite enzyme would be designed.Molecules expressed in erythrocytic stage of Babesia caballi and B.equi were also characterized. Libraries of cDNAs from B.caballi and B.equi merozoites were screened with infected horse sera. From B.equi cDNA library, two genes encoding a glutamic acid-rich protein (GARP) and ribosomal protein (L10) were obtained. GARP contained motifs rich in glutamic acid which showed structural similarity to surface molecules expressed in intraerythrocytic stage of malaria parasites. A gene for B.caballi rhoptry-associated protein-l (RAP-1) , one of parasite proteins which plays a central role in parasite invasion into host erythrocytes was also cloned.N-terminal amino acid sequences of veil proteins of T.sergenti were determined and compared to sequences of known erythrocyte proteins. As no homologous poplypeptides were found in data base search, veil proteins were concluded to be parasite-origin.