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Molecular characterization of a third malic enzyme-like AP65 adhesin gene of Trichomonas vaginalis
J L O'Brien1, C M Lauriano, J F Alderete
1Department of Microbiology, University of Texas Health Science Center, San Antonio 78284-7758, USA.
Abstract:
Adherence to the vaginal epithelium by the sexually transmitted parasite Trichomonas vaginalis is mediated by four trichomonad surface proteins (AP65, AP51, AP33 and AP23). We recently showed that the 65-kDa adhesin is a member of a multigene family comprised of two similar but distinct proteins, AP65-1 and AP65-2, encoded by the genes ap65-1 and ap65-2, respectively. An additional immuno-crossreactive clone, the 1.2 kb F11.1 cDNA, was isolated from a phagemid expression library and encoded a fusion protein of approximately 46,000 daltons (46 kDa) that bound to HeLa cell surfaces. A significant portion of the 5' end was missing which, using the 5'-RACE method, was obtained and combined with the F11.1 clone to give a full-length cDNA. The ap65-3 gene encoded for a protein of 567 amino acids with a molecular mass of 63.1 kDa. The gene showed 88% and 96% identity at the DNA level with ap65-1 and ap65-2, respectively. Restriction mapping confirmed that the three AP65 genes are different. Southern analysis revealed that the ap65-3 gene is present in the T. vaginalis genome in multiple copies. Experiments with agar clones of trichomonads showed that each gene of the multigene family is present in all parasites, and Northern analysis showed that ap65-3 is expressed and transcriptionally regulated by iron. The ap65-3 gene had a leader sequence and, as with ap65-1 and ap65-2, showed significant homology to malic enzyme. Finally, analysis of the 3'-untranslated regions revealed that the transcript of ap65-3 had a long poly (A) tail in comparison to ap65-1 and ap65-2. Even more intriguing, sequences were found that may relate to differential degradation of select AP65 transcripts, such as the sequence motifs AUUUA for ap65-1 mRNA and UUAUUUAU for the ap65-2 mRNA, which were not found for ap65-3.
Insights
Trichomonas vaginalis uses AP65 adhesins for vaginal epithelium adherence. A newly identified ap65-3 gene, part of a multigene family, is expressed and regulated by iron, showing distinct transcript features.
Area of Science:
- Molecular Parasitology
- Microbial Pathogenesis
- Genomics and Transcriptomics
Background:
- Adherence to the vaginal epithelium by Trichomonas vaginalis is crucial for infection, mediated by surface proteins like AP65.
- The AP65 adhesin family was previously known to include AP65-1 and AP65-2, encoded by distinct genes.
- Understanding the full AP65 gene family is essential for elucidating parasite-host interactions.
Purpose of the Study:
- To identify and characterize novel genes within the AP65 adhesin multigene family.
- To investigate the expression and regulation of the newly identified AP65 gene.
- To compare the genetic and transcriptomic features of different AP65 family members.
Main Methods:
- Isolation and sequencing of a full-length cDNA using 5'-RACE and existing clones (F11.1).
- Gene identification and characterization through restriction mapping, Southern analysis, and Northern analysis.
- Sequence homology analysis (DNA and protein) and 3'-untranslated region analysis.
Main Results:
- A new gene, ap65-3, was identified, encoding a 63.1 kDa protein with 88-96% DNA identity to ap65-1 and ap65-2.
- Southern analysis confirmed multiple copies of ap65-3 in the T. vaginalis genome; Northern analysis showed its expression is iron-regulated.
- ap65-3 shares homology with malic enzyme and possesses a long poly (A) tail, with distinct 3'-UTR sequences compared to ap65-1 and ap65-2, suggesting differential mRNA stability.
Conclusions:
- The AP65 adhesin family in T. vaginalis is more complex than previously thought, including at least three distinct genes.
- The ap65-3 gene is transcriptionally regulated by iron availability, a key environmental factor for the parasite.
- Differential regulation of AP65 transcripts, potentially through 3'-UTR sequences, may control protein levels and parasite adherence.