Molecular characterization and expression of a N-acetylneuraminate lyase gene from Trichomonas vaginalis.
|Title:||Molecular characterization and expression of a N-acetylneuraminate lyase gene from Trichomonas vaginalis.|
|Authors:||Meysick, Karen C.|
|Abstract:||By screening a Trichomonas vaginalis $\lambda$gt11 cDNA library with anti-serum raised against a purified preparation of T. vaginalis cell-detaching factor (CDF), two clones were identified. Molecular analysis of the cDNA sequences indicated that the clones were distinct and could potentially represent two unique T. vaginalis genes. Further characterization of the CDF anti-serum used in the initial library screening indicated that the serum possessed reactivity to CDF and a variety of other T. vaginalis proteins. The multiple specificities of the anti-serum are likely to have contributed to the identification of the two distinct cDNA clones. While the relationship between the weaker of the two immunoreactive cDNA sequences (CDF-2) and the CDF protein was unclear, the results of Northern hybridization and sequence analysis suggested that the strongly reactive CDF-1 clone did not contain sequences representing a CDF gene. Since the CDF-1 cDNA clone contained an almost complete coding sequence and because the encoded polypeptide exhibited significant protein sequence similarity to bacterial N-acetylneuraminate (Neu5Ac) lyases, this T. vaginalis sequence was further analyzed. Using the cDNA sequence and cloned segments of overlapping genomic DNA, the complete coding and flanking regions of this T. vaginalis gene, designated TvnanA, were determined. TvnanA is a single copy gene that lacks introns and contains a 954 bp open-reading frame that is predicted to encode a 318 amino acid polypeptide with a calculated molecular mass of 35 kDa. The gene is transcribed as a single 1.1 kb polyadenylated mRNA with short 5$\sp\prime$ (17-18 nt) and 3$\sp\prime$ (28 nt) untranslated regions. A sequence resembling the T. vaginalis initiator element was identified in the region surrounding the transcriptional initiation site, however this Inr-like element did not direct accurate gene transcription. The TvnanA encoded polypeptide shows 35% identity and 53% similarity to the Neu5Ac lyase of Escherichia coli, and 73% identity to a predicted Neu5Ac lyase of Haemophilus influenzae. The homology includes significant similarity within a region corresponding to the putative active site pocket of the E. coli enzyme. A complete copy of the TvnanA gene was assembled and the gene product was expressed in E. coli as a glutathione S-transferase fusion protein. In the absence of detergents required for efficient solubilization, the T. vaginalis gene product exhibited Neu5Ac lyase activity that was 30 times greater than background activity in controls. Lyase activity was increased a further 5-fold by deletion of the N-terminal hydrophobic region of the T. vaginalis protein. While fusion proteins exhibited Neu5Ac lyase activity, they did not appear to be capable of producing cell-detaching activity when incubated in the presence of eukaryotic cell monolayers. Preliminary experiments to determine the size and location of the T. vaginalis Neu5Ac lyase suggests that the enzyme is present as an intracellular 35 kDa species. While questions remain concerning the regulation of the TvnanA gene and the role of the N-terminal region in the encoded polypeptide, this is the first reported characterization of a T. vaginalis gene product involved in sialic acid metabolism. The presence of this enzyme combined with the reports of a T. vaginalis neuraminidase suggests that the parasite may utilize host sialic acid as a nutrient source.|
|Collection||Thèses, 1910 - 2010 // Theses, 1910 - 2010|