Cloning and molecular characterization of two genes encoding adhesion proteins involved in Trichomonas vaginalis cytoadherence

Abstract

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.

Characterization of an Iron-responsive Promoter in the Protozoan Pathogen Trichomonas vaginalis

“…2B). These findings are consistent with previous results describing the expression features of the ap65 gene family in other T. vaginalis isolates (8,12,15) and show that our experimental system is suitable for the study of iron-regulated expression of the ap65-1 gene.…”

“…Iron, which is an essential nutrient for almost every organism, is particularly important for T. vaginalis as it regulates growth rate, metabolic activities, and the expression of certain virulence phenotypes such as cytoadherence and resistance to complement lysis (6 -11). At the molecular level, iron has been shown to up-regulate the expression of a number of cellular proteins including a group of putative adhesin molecules, the identities of which are still controversial (8,9,(12)(13)(14)(15)(16)(17)(18)(19). Iron has also been shown to regulate the phosphorylation level of a major surface immunogen P270, which may be responsible for immune evasion (20).…”

“…In this study, the ap65-1 gene, which encodes a 65-kDa protein reputed to be one of the surface adhesin proteins (12), was selected as a model system to study iron-induced gene expression in T. vaginalis. The DNA regulatory elements in the ap65-1 promoter were characterized by promoter analysis in vivo in conjunction with DNA-protein interaction assays in vitro.…”

Host and Tissue Specificity of Trichomonas vaginalis Is Not Mediated by Its Known Adhesion Proteins

“…Moreover, two of the four adhesins, AP65 and AP33, have been characterized, and their sequences are known (6,14,25). As can be inferred from their protein sequences, which have a striking homology with the hydrogenosomal enzymes malic dehydrogenase (decarboxylating) (2,6,15,22,25) and succinyl coenzyme A synthetase (14,23), respectively, these proteins could possess an intrinsic affinity for membranes. On the basis of these data, we can suppose that the use of solubilized proteins, instead of live intact parasites, as well as the use of trypsinized and glutaraldehyde-fixed epithelial cells, is not a more appropriate technique to detect adhesins.…”

“…In the past, several reports have described the molecular characterization of the different steps involved in interaction of the parasite with the human vaginal epithelial cells. Adhesion of the parasite is thought to be mediated by four trichomonad surface proteins, reportedly AP65, AP51, AP33, and AP23 (5,6), which are believed to recognize specific proteins of the host by a ligand-receptor type of interaction (3,7,8), although the receptors in the host cell have not yet been identified. The putative role of these proteins in adhesion has been characterized by using HeLa cells, because of their epithelial nature and the genital epithelium origin.…”