Tomoyoshi Nozaki

Molecular cloning and characterization of the genes encoding two isoforms of cysteine synthase in the enteric protozoan parasite Entamoeba histolytica

The enteric protozoan parasite Entamoeba histolytica was shown to possess cysteine synthase (CS) activity. The cDNA and genomic clones that encode two isoforms of the E. histolytica CS were isolated and characterized from a clonal strain of E. histolytica by genetic complementation of the cysteine-auxotrophic Escherichia coli NK3 with an E. histolytica cDNA library. The two types of the E. histolytica CS genes differed from each other by three nucleotides, two of which resulted in amino acid substitution. Deduced amino acid sequences of the E. histolytica CS, with a calculated molecular mass of 36721 Da and an isoelectric point of 6.39, exhibited 38-48% identity with CS of bacterial and plant origins. The absence of the amino-terminal transit peptide in the deduced protein sequences and the presence of the CS protein mainly in the supernatant fraction of the amoebic lysate after cellular fractionation suggested that the identified E. histolytica CS genes encoded cytosolic isoforms. Substrate specificity of the recombinant E. histolytica CS was similar to that of plant CS. Phylogenetic analysis indicates that the amoebic CS, first described in Protozoa, does not belong to any families of the CS superfamily, and represents a new family.

Purification and identification of major soluble 40-kDa antigenic protein from Entamoeba histolytica: its application for serodiagnosis of asymptomatic amebiasis

One of the major soluble antigenic proteins of Entamoeba histolytica was purified to homogeneity and identified on a molecular basis. Its recombinant protein was expressed in Escherichia coli as a fusion protein with Shistosoma japonicum glutathione S-transferase. Apparent molecular weight of the purified antigenic protein was estimated to be 40-kDa and molecular-based analysis indicated that the purified protein was NADP+-dependent alcohol dehydrogenase (EhADH1). The application of the purified protein for the serodiagnosis of amebiasis was evaluated using an enzyme-linked immunosorbent assay applied to sera obtained from patients with amebiasis and healthy human controls. The purified protein was well recognized by the sera from asymptomatic amebiasis humans (22/22, 100%), whereas, it was less recognized by the sera from symptomatic amebiasis patients (5/16, 31%) with amebic colitis or liver abscess. To confirm the antigenicity of EhADH1, the recombinant glutathione S-transferase-EhADH1 fusion protein was also evaluated by the enzyme-linked immunosorbent assay using the same sera. The recombinant protein was also recognized by the sera from asymptomatic amebiasis humans (14/22, 64%) and less recognized by the sera from symptomatic amebiasis patients (2/16, 13%). These results suggest that the purified protein is applicable antigen for serodiagnostic screening of asymptomatic amebiasis humans.

Codon usage in Entamoeba histolytica, E. dispar and E. invadens

Abstract We analyzed the frequencies of genetic codon usage of 68 non-redundant protein coding genes from the human-pathogenic E. histolytica (28 117 codons), 6 from the non-pathogenic E. dispar (1744 codons), and 4 from the reptilian E. invadens (933 codons). The A + U contents of the protein coding sequences from E. histolytica , E. dispar , and E. invadens were 67%, 66%, and 58%, respectively. The nucleotide frequency in the third position was strongly biased toward A + U in E. histolytica and E. dispar (85% and 82%, respectively); the degree of the A + U bias was higher in the third position than that in the first or second position. In contrast, the nucleotide frequency in the third position was less biased in E. invadens (60% A + U) than in E. histolytica and E. dispar . Codon usage was biased in accordance with the A + U preference in the third position in E. histolytica and E. dispar . However, no apparent difference in the codon usage was found between E. histolytica and E. dispar . The codon usage in E. invadens was found less biased; the nucleotide biases observed in the third position of the synonymous codons for several amino acids including leucine, tyrosine, cysteine, and histidine of the E. histolytica and E. dispar genes were reversed or absent. The codon usage in Entamoeba species significantly differed from that in other amitochondrial protist, Giardia lamblia and Trichomonas vaginalis. Two sequences encoding ribosomal protein S10 and S27 showed significantly smaller codon biases than the rest of E. histolytica sequences, suggesting that these ribosomal proteins might be under specific functional constraint of codon usage. The differences in the A + U content of the coding sequences and in the codon usage between the mammalian E. histolytica and E. dispar and the reptilian E. invadens suggested that the reptilian Entamoeba species were distantly related to the mammalian species. These results may aid in elucidating pressures that facilitate changes in the patterns of the genetic codon usage.