Cloning and molecular characterization of two genes encoding adhesion proteins involved in Trichomonas vaginalis cytoadherence.
Cytoadherence to the vaginal epithelium is a critical step in infection by the eukaryotic flagellate Trichomonas vaginalis. Four trichomonad surface proteins (AP65, AP51, AP33 and AP23) mediate cytoadherence. The cDNA encoding the AP65 adhesin was isolated from a phagemid cDNA expression library by screening with antiserum and monoclonal antibody (mAb) raised against the purified trichomonad AP65 protein. Two clones, F11.2 and F11.5, coded for immuno-crossreactive recombinant proteins that possessed functional properties equal to the T. vaginalis AP65 adhesin. Analysis of full-length sequences corresponding to the F11.2 and F11.5 cDNAs revealed that both contained 1701-base open reading frames (ORFs) that encoded proteins of 63 281 daltons and 83 087 daltons, respectively. Comparison of the full-length sequences showed 87% identity at the nucleotide level and 91% identity at the protein level. Restriction-enzyme mapping and Southern analysis reaffirmed the distinctness of the F11.2 and F11.5 cDNAs, indicating that two different AP65 genes (now called ap65-1 and ap65-2) are present in the T. vaginalis genome in at least two copies each. Northern analysis detected high levels of transcript of approximately 1.8 kb for both ap65-1 and ap65-2 genes in trichomonads grown only in high-iron medium, confirming the transcriptional regulation of adhesin synthesis by iron. Homology searches revealed significant similarity (38% amino acid identity and 54% nucleotide identity) to malic enzymes. However, purified malic enzyme and mAb to AP65 crossreactive with malic enzyme neither inhibited cytoadherence of T. vaginalis to host cells nor prevented binding of the trichomonad AP65 to HeLa cells in a ligand assay.
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002448:Cell Adhesion / Q000235:genetics; D015815:Cell Adhesion Molecules; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D017125:Genes, Protozoan / Q000235:genetics*; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D007501:Iron / Q000494:pharmacology*; D008969:Molecular Sequence Data; D005810:Multigene Family / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis; D016053:RNA, Protozoan / Q000096:biosynthesis; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D014158:Transcription, Genetic / Q000187:drug effects; D014246:Trichomonas vaginalis / Q000235:genetics*
C096723:AP65-1 protein, Trichomonas vaginalis; C096724:AP65-2 protein, Trichomonas vaginalis; D015815:Cell Adhesion Molecules; D018076:DNA, Complementary; D015800:Protozoan Proteins; D012333:RNA, Messenger; D016053:RNA, Protozoan; D011993:Recombinant Fusion Proteins; D007501:Iron