A novel surface protein of Trichomonas vaginalis is regulated independently by low iron and contact with vaginal epithelial cells

Abstract

Background: Trichomonosis caused by Trichomonas vaginalis is the number one, non-viral sexually transmitted disease (STD) that affects more than 250 million people worldwide. Immunoglobulin A (IgA) has been implicated in resistance to mucosal infections by pathogens. No reports are available of IgA-reactive proteins and the role, if any, of this class of antibody in the control of this STD. The availability of an IgA monoclonal antibody (mAb) immunoreactive to trichomonads by whole cell (WC)-ELISA prompted us to characterize the IgA-reactive protein of T. vaginalis.

Results: An IgA mAb called 6B8 was isolated from a library of mAbs reactive to surface proteins of T. vaginalis. The 6B8 mAb recognized a 44-kDa protein (TV44) by immunoblot analysis, and a full-length cDNA clone encoded a protein of 438 amino acids. Southern analysis revealed the gene (tv44) of T. vaginalis to be single copy. The tv44 gene was down-regulated at both the transcriptional and translational levels in iron-depleted trichomonads as well as in parasites after contact with immortalized MS-74 vaginal epithelial cells (VECs). Immunofluorescence on non-permeabilized organisms confirmed surface localization of TV44, and the intensity of fluorescence was reduced after parasite adherence to VECs. Lastly, an identical protein and gene were present in Tritrichomonas foetus and Trichomonas tenax.

Conclusion: This is the first report of a T. vaginalis gene (tv44) encoding a surface protein (TV44) reactive with an IgA mAb, and both gene and protein were conserved in human and bovine trichomonads. Further, TV44 is independently down-regulated in expression and surface placement by iron and contact with VECs. TV44 is another member of T. vaginalis genes that are regulated by at least two independent signaling mechanisms involving iron and contact with VECs.

Figure 1

T. vaginalis surface immunofluorescence by monoclonal antibody (mAb) 6B8 and immunoblot detection of a trichomonad protein. A. Fluoresceine isothiocyanate- conjugated goat anti-IgA antibody reacts with the surface of non-permeabilized T. vaginalis (A1), T. foetus (A2), and T. tenax (A3) incubated with IgA mAb 6B8. Brightfield microscopy is provided below the fluorescence panels to illustrate the integrity of the organisms. Fluorescence experiments were performed at least 5 times, and the distinct patchy pattern of fluorescence for T. tenax contrasted with the similar overall surface fluorescence for T. vaginalis and T. foetus. B. Total proteins of 10⁷ trichomonads of T. vaginalis (lane 1), T. foetus (lane 2), and T. tenax (lane 3) were separated on 10% polyacrylamide gels by SDS-PAGE prior to blotting onto Hybond-P™ membranes. Blots were probed with mAb 6B8. Molecular weight of the protein is indicated in kilodaltons (kDa) (×1000).

Figure 2

TV44 amino acid sequence alignment with proteins of BLAST search. The corresponding proteins of Entamoeba histolytica [XM 645307.1], Saccharomyces cerevisiae [P 53040], Arabidopsis thaliana [AY463621.1], Homo sapiens [AAH18115], and Candida albicans [XM 711022.1] were aligned by the Clustal W program. The conserved residues are indicated by black shading, and similarity is indicated by grey shading. TV44 had partial homology to transcription initiation factor (TFIID) of E. histolytica (13% identity) and S. cerevisiae (16%). TV44 had homology to the TATA-binding protein associated factor of A. thaliana (15%) and H. sapiens (13%) and, in addition, to the RNA pol II transcription factor of C. albicans (17%).

Figure 3

Southern blot analysis of T. vaginalis genomic DNA. Ten micrograms of genomic DNA was digested with restriction enzymes before separating on 0.9% agarose gels and blotting onto Hybond™ -N+ membranes. The membranes were then probed with a labeled PCR product containing the coding region of the tv44 gene. Numbers on the left show the positions of a 1-kilobase (kb) ladder.

Figure 4

The tv44 gene expression is down-regulated by low iron. A. Ethidium bromide-stained agarose gels after electrophoresis of RT-PCR products for the tv44 transcript in parasites grown in normal medium (lane 1) or in low- (lane 2) versus high-iron (lane 3) medium. The bottom panel shows the RT-PCR products for the T. vaginalis α-tubulin (α-tub) gene as a control to show equal amounts of RNA in the PCR reactions. B. Immunoblot detection by mAb 6B8 of a 44-kDa protein after SDS-PAGE and blotting as described in the legend of Figure 1 of total proteins derived from trichomonads grown in normal medium (lane 1) and high- (lane 2) versus low-iron (lane 3) medium, as indicated. Numbers on the right are molecular weight standards in kilodaltons (× 1000).

Figure 5

Surface expression of TV44 on trichomonads decreases after contact with VECs. FITC-conjugated goat anti-IgA antibody bound to IgA mAb 6B8 on the surface of non permeabilized T. vaginalis organisms as evident by uniform fluorescence seen in panel B1. In contrast, parasites isolated after contact with MS-74 VECs and handled identically had little or no detectable fluorescence as seen in panel B2. Brightfield pictures show the integrity of parasites used for the assay. No fluorescence was detectable in the absence of the mAb 6B8 as negative controls (not shown).

Figure 6

T. vaginalis contact with VECs results in decreased tv44 transcription (A and C) and amounts of TV44 (B and D). A. Decreased amounts of tv44 RT-PCR products as evidenced by ethidium bromide-stained gels after electrophoresis in 1% agarose from trichomonads after contact with immortalized MS-74 VECs (lane 2) compared to parasites without contact to VECs (lane 1). The equal quantities of the α-tubulin (α-tub) RT-PCR products show equal amounts of RNA in the PCR reactions. B. Decreased amounts of TV44 detected by mAb 6B8 on immunoblots of total proteins of trichomonads after contact with VECs (lane 2) compared to control organisms (lane 1). Proteins for SDS-PAGE were from equal numbers of organisms as described in the legend of Figure 1C and D. The relative amounts of the tv44 gene RT-PCR product in stained agarose gels (A) and TV44 on immunoblots (B) were obtained by scanning the bands from parts A and B using the Scion image beta program. The amounts of PCR product and protein obtained from parasites without contact with VECs was normalized to 100% (bars1) for comparison with PCR product of organisms after contact with host cells (bars 2).