Epitopes of the highly immunogenic Trichomonas vaginalis α-actinin are serodiagnostic targets for both women and men

There is a need for a point-of-care serodiagnostic test for women and men for sexually transmitted infections (STIs) caused by Trichomonas vaginalis. Sera from women with this STI and sera from men that were analyzed in studies showing a relationship between serostatus and prostate cancer are highly seropositive in response to trichomonad α-actinin and its truncated protein (ACT-P2) (positive control sera). Epitope mapping experiments showed that positive control sera from women had antibodies to 13 distinct epitopes, 5 of which were detected by positive control sera from men. Sera from women and men that were unreactive with α-actinin (negative control sera) failed to detect any of the epitopes or other α-actinin amino acid sequences. The T. vaginalis α-actinin amino acid sequence and the sequences of the epitopes showed little or no identity with those of other proteins of microbial pathogens or the human α-actinin 1 (HuACTN1) homolog. Immunoassays such as dot blot, immunoblot, and enzyme-linked immunosorbent assays were used. Positive control sera did not detect HuACTN1 in immunoassays, and the range of levels of identity of α-actinin epitopes with HuACTN1 was 0% to 50%. Comparison of the T. vaginalis α-actinin epitopes with proteins in data banks, such as Tritrichomonas suis, Candida albicans, and Saccharomyces cerevisiae proteins, gave a range of identity levels of 0% to 22%. Specific 15-mer peptide epitopes of α-actinin with low to no identity with other proteins were synthesized and were reactive with positive control sera only. These findings identify epitopes of α-actinin as candidate serodiagnostic targets and suggest strongly that a highly seropositive reaction to α-actinin suggests exposure to T. vaginalis

Demonstration of elevated amounts of PIM1 and HMGA1 proteins in PECs after adherence by <i>T. vaginalis</i> (<i>Tv</i>).

<p>In this experiment, trichomonads were added to T25 confluent monolayers of PECs (lane labeled+) using a parasite to PEC ratio of 10∶1. PECs without added organisms are labeled with a minus sign (−). This ratio of 10∶1 was chosen because it has been shown to yield at least one parasite attached per epithelial cell in adherence assays and to optimally signal epithelial cells for up-regulation of expression of genes <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002801#ppat.1002801-Kucknoor1" target="_blank">[13]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002801#ppat.1002801-Garcia1" target="_blank">[27]</a>. After visible attachment to PECs, non-adherent organisms were decanted and fresh PEC medium added followed by incubation at 37°C for an additional 30 min. The flask was then placed directly in ice for detachment of organisms, after which PECs were washed and removed from the flask for preparation of total proteins for immunoblotting using established protocols, polyclonal rabbit antibodies produced in our laboratories, and equal loading of protein onto gels, as detailed previously <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002801#ppat.1002801-Hu1" target="_blank">[28]</a>. Under conditions of exposure of PECs with or without <i>T. vaginalis</i>, no change in the amount of other cellular proteins was detected, as evidenced by no changes in the amounts of Akt and Bad, and this served to show equal amounts of total proteins loaded onto gels for SDS-PAGE and immunoblotting <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002801#ppat.1002801-Hu1" target="_blank">[28]</a>. Prebleed rabbit serum was used as the negative control and gave no reactivity.</p