Adherence to human vaginal epithelial cells signals for increased expression of Trichomonas vaginalis genes

Abstract

Host parasitism by Trichomonas vaginalis is complex, and the adhesion to vaginal epithelial cells (VECs) by trichomonads is preparatory to colonization of the vagina. Since we showed increased synthesis of adhesins after contact with VECs (A. F. Garcia, et al., Mol. Microbiol. 47:1207-1224, 2003) and more recently demonstrated up-regulated gene expression in VECs after parasite attachment (A. S. Kucknoor, et al., Cell. Microbiol. 7:887-897, 2005), we hypothesized that enhanced expression of adhesin and other genes would result from signaling of trichomonads following adherence. In order to identify the genes that are up-regulated, we constructed a subtraction cDNA library enriched for differentially expressed genes from the parasites that were in contact with the host cells. Thirty randomly selected cDNA clones representing the differentially regulated genes upon initial contact of parasites with host cells were sequenced. Several genes encoded functional proteins with specific functions known to be associated with colonization, such as adherence, change in morphology, and gene transcription and translation. Interestingly, genes unique to trichomonads with unknown functions were also up-regulated. Semiquantitative reverse transcription-PCR (RT-PCR) confirmed expression of select genes. An increased amount of protein was demonstrated by immunoblotting with monoclonal antibody. Finally, we showed the transcriptional regulation of some genes by iron by using RT-PCR. To our knowledge, this is the first report addressing the differential regulation of T. vaginalis genes immediately upon contact with VECs.

FIG. 1.

RNA isolation and PCR analysis of subtraction efficiency. (A) Total RNA was isolated from primed parasites after contact with VECs (pTv) and control organisms handled identically (T. vaginalis [Tv]). Total RNA (3 μg) was separated on a 1.2% agarose gel followed by staining with EtBr to visualize the purity and assess degradation of RNA. 28S and 18S refer to the rRNA bands of the VECs and the parasites. (B) PCR using α-tubulin primers was performed on unsubtracted and subtracted cDNAs. Aliquots (5 μl) were removed at a predetermined number of cycles and analyzed by EtBr-stained gels after electrophoresis in 1.2% agarose. The α-tubulin product appeared only after 25 cycles in the subtracted sample compared to 15 cycles in the unsubtracted sample.

FIG. 2.

Representative experiment showing confirmation of gene expression patterns in primed (pTv) and control (T. vaginalis [Tv]) parasites by semiquantitative RT-PCR analyses. Total RNA from pTv and T. vaginalis was isolated as detailed in Materials and Methods. RNA was reverse transcribed using oligo(dT) primer, and PCR was performed using gene-specific primers (Table 1). (A) RT-PCR products separated on EtBr-stained gels after electrophoresis in 2% agarose. (B) Gene expression pattern relative to the housekeeping trichomonad α-tubulin gene used as a control. The values were obtained by scanning the intensities of bands from pictures of the gels in A using the Scion image beta program. The expression for each gene was relative to baseline density for α-tubulin plotted on the graph. This experiment was repeated on four separate occasions with similar results.

FIG. 3.

Detection of protein expression in T. vaginalis upon adherence to VECs. Cell lysates were prepared from equivalent numbers of primed (pTv) and control (T. vaginalis [Tv]) parasites prior to SDS-PAGE and blotting onto nitrocellulose membranes, as described in Materials and Methods. Quadruplicate blots were probed with the MAbs F5, DM116, HA423, and B512, specific for AP33, AP65, α-actinin, and α-tubulin, respectively. The numbers indicated on the left-hand side are the molecular mass standards. A negative control without MAb and with only secondary antibody was always nonreactive in duplicate blots (data not shown).

FIG. 4.

Representative experiment showing the effect of iron on regulation of selected genes. Total RNA from parasites grown in normal TYM medium (N), iron-depleted medium (L), and iron-replete medium (H) was used in RT-PCR. (A) PCR products were separated on 2% agarose gels for visualization after EtBr staining. (B) Bar graph plotted using the relative values of band intensity obtained by Scion image beta scanning. Again, all values were normalized to the band intensity of α-tubulin as a control. This experiment was repeated on four separate occasions with similar results.