Antisense RNA decreases AP33 gene expression and cytoadherence by T. vaginalis

Abstract

Background: Host parasitism by Trichomonas vaginalis is complex. Adherence to vaginal epithelial cells (VECs) is mediated by surface proteins. We showed before that antisense down-regulation of expression of adhesin AP65 decreased amounts of protein, which lowered levels of T. vaginalis adherence to VECs. We now perform antisense down-regulation of expression of the ap33 gene to evaluate and confirm a role for AP33 in adherence by T. vaginalis. We also used an established transfection system for heterologous expression of AP33 in T. foetus as an additional confirmatory approach.

Results: We successfully select stable trichomonads with sense (S) and antisense (AS) plasmids. RT-PCR confirmed decreased amounts of ap33 mRNA in AS-transfected parasites, and decreased amounts of AP33 had no effect on growth and viability when compared to wild-type (wt) trichomonads. Immunoblots of proteins from AS-transfectants gave significant decreased amounts of functional AP33 capable of binding to host cells compared to wt- and S-transfected trichomonads. As expected, AS-transfectants had lower levels of adherence to VECs, which was related to reduction in surface expression of AP33. Stable expression of T. vaginalis AP33::HA fusion in T. foetus was confirmed by immunoblots and fluorescence. The episomally-expressed surface AP33::HA fusion increased adherence of trichomonads to human VECs, which was abrogated with anti-AP33 serum.

Conclusion: These results using both antisense inhibition of gene expression and AP33 synthesis and the heterologous expression of AP33 in T. foetus confirms a role for this protein as an adhesin in T. vaginalis.

Figure 1

Transfection and RT-PCR showing reduced ap33 mRNA levels in T. vaginalis trichomonads transfected with the antisense plasmid. Part A shows PCR amplification of the neo coding region in transfected parasites. The ethidium bromide (EtBr)-stained band after electrophoresis in 1% agarose is the PCR product of the neo gene that was amplified using DNA from transfected T. vaginalis parasites. As expected, no PCR product was expected from wt organisms, and a predicted product was obtained from the plasmid used directly during PCR as a control (part A, lane labeled C). Part B gives bands after electrophoresis as in part A showing RT-PCR products for the ap33 S (panel a) and AS transcript (panel b). The RT-PCR product for ap65and α-tubulin are presented in panels c and d, respectively. Part C illustrates the quantitation of the transcript bands in part B (panel a) for the ap33 transcript in AS-transfected trichomonads compared to S-transfected and wt parasites. The bar graph shows the relative amounts of RT-PCR products for ap33. The amount of wild type ap33 transcript was normalized 100%. Quantification was done following Scion image β program.

Figure 2

Immunoblot analysis of S- and AS-transfected trichomonads and wt T. vaginalis organisms. Part A presents triplicate blots of total protein from 10⁷ trichomonads after SDS-PAGE on 10% acrylamide before blotting onto Hybond-P membranes. The blots were probed with mAb F5.2 to AP33 (panel 1), mAb 12G4 to AP65 (panel 2) [17], and mAb to α-tubulin (panel 3). Part B presents immunoblot results from a ligand assay, which was performed using extracts from equal numbers of organisms.

Figure 3

Representative growth kinetics of wt organisms and S- and AS-transfected trichomonads cultivated in batch culture. Medium was inoculated with 10⁵ parasites, and cell densities at different time points were enumerated using a hemocytometer. Similar results were obtained from three growth experiments performed independently at different days.

Figure 4

Immunofluorescence showing decreased surface expression of AP33 in non-permeabilized, AS-transfected (panel C) compared to S-transfected (panel B) and wt trichomonads (panel A). Rabbit polyclonal anti-AP33 serum characterized previously [24] was used as described in Methods.

Figure 5

Relative adherence of AS-transfectants (bar AS) compared with wt-(bar wt) and S-transfected (bar S) parasites. T. vaginalis parasites expressing antisense display decreased adherence to MS-74 VECs. The extent of adherence by wt organisms was normalized to 100% for comparative purposes. The results are the average from four different experiments. Each experiment was performed in quadruplicate samples. The asterisk illustrates that this extent of decrease was statistically significant (p < 0.05).

Figure 6

Immunoblot and fluorescence analyses showing episomal expression of AP33::HA in T. foetus. A and B: Total protein from 10⁷ parasites were separated on 10% SDS -PAGE and blotted on to Hybond-P membrane. The blots were probed with mAb F 5.2 to AP33 (A) and mAb to HA (B). C: Immunofluorescence was performed using anti-HA mAb on non-permeabilized wt (Tf) and transfected trichomonads (Tf-AP33::HA). Brightfield microscopy shows the same trichomonads in the field used for fluorescence microscopy.

Figure 7

T. foetus with surface AP33::HA displays enhanced level of adherence to immortalized MS-74 VECs. Transfected trichomonads with AP33::HA (bar labeled Tf-AP33) had higher levels of adherence compared to wt T. foetus (bar Tf), as before for heterologously-expressed AP65 [26]. The percent level of adherence was adjusted to that seen for T. vaginalis (Tv). Trichomonads expressing AP33::HA were pretreated with anti-AP33 antiserum (bar Tf-AP33 + antibody). The results are the average from four different experiments, and each experiment was carried out using quadruplicate samples.