Only two of the Trichomonas vaginalis triplet AP51 adhesins are regulated by iron.

The sexually transmitted parasite Trichomonas vaginalis cytoadheres to the vaginal epithelium, and four candidate trichomonad adhesins have been identified. One such protein, termed AP51, was characterized further. To do this, we studied a 1 kb cDNA clone (AP51.2) isolated from a phagemid expression library, which encoded a fusion protein of approximately 38 kDa that was immuno-crossreactive with anti-AP51 serum and retained functional adhesive properties. We performed 5'-PCR amplification to recover the missing 5' end in order to provide the complete cDNA sequence for the gene encoded by AP51.2 (ap51-2). Other PCR products revealed almost complete sequences for two additional ap51 genes, making AP51 a member of a multigene family of at least three distinct proteins and genes. The ap51-1 and ap51-3 genes each encoded for 407 amino acids while ap51-2 encoded 408 amino acids, and not unexpectedly, these genes had a high percent identity at the DNA and amino acid levels. Mapping confirmed the sequence distinctions and uniqueness of the three ap51 genes. Southern analysis using gene-specific probes revealed the single copy nature of each of the ap51 genes, all of which were present among the numerous agar clones of single trichomonads of the isolates tested. Importantly, Northern analysis showed transcriptional regulation by iron of only the ap51-1 and ap51-3 genes but not ap51-2, perhaps indicating the presence of two bona fide isoforms of the ap51 genes. The 3'-untranslated region of ap51-3 had a short poly (A) tail as well as the sequence motif AUUUA, which may relate to differential degradation of ap51-3 transcripts, in comparison to ap51-1 and ap51-2. Finally, the ap51 genes had partial homology to the beta-subunit of succinyl-CoA synthetase, reinforcing the idea that molecular mimicry may play a role in host parasitism by T. vaginalis.

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.