The transcription efficiency of an adhesion protein gene, ap65-1, in Trichomonas vaginalis varies with changes in the iron supply and with the growth stage. In the present study, two Myb recognition elements, MRE-1/ MRE-2r and MRE-2f, were found to play antagonistic roles in regulating the iron-inducible activity of an ap65-1 reporter gene. Intriguingly, either of these elements was shown to be sufficient to repress basal activity, but together they were also shown to activate growth-related activity of the reporter gene in iron-depleted cells. A myb1 gene which encodes a 24-kDa protein containing a Myb-like R2R3 DNA binding domain was identified from Southwestern screening of MRE-2f-binding proteins. The Myb1 protein was detected as a major 35-kDa protein which exhibited variations in nuclear concentration with changes in the iron supply. A recombinant Myb1 protein was shown to differentially interact with MRE-1/MRE-2r and MRE-2f in vitro. Overexpression of hemagglutinin-tagged Myb1 in T. vaginalis resulted in repression or activation of ap65-1 transcription in iron-depleted cells at an early and a late stage of cell growth, respectively, while iron-inducible ap65-1 transcription was constitutively repressed. The hemagglutinin-tagged Myb1 protein was found to constantly occupy the chromosomal ap65-1 promoter at a proximal site, but it also selected two more distal sites only at the late growth stage. Together, these observations suggest that Myb1 critically regulates multifarious ap65-1 transcription, possibly via differential selection of multiple promoter sites upon environmental changes.As the most common sexually transmitted disease of nonviral origin in humans, trichomoniasis caused by infection with the protozoan parasite Trichomonas vaginalis is an important risk factor for the transmission of human immunodeficiency virus (33). Along with increasing numbers of drug-resistant clinical T. vaginalis isolates (7, 9), trichomoniasis is emerging as a major threat to public health.Iron availability, which periodically varies in the human vagina, where the parasite colonizes, regulates the cytoadherence of T. vaginalis, possibly through controlling the expression of several adhesion proteins in transcription initiation and protein trafficking steps (2,11,18,35). Although only a few T. vaginalis genes have been characterized in detail, the most common example seems to suggest that T. vaginalis uses a conserved initiator (Inr) sequence as the sole core promoter element to regulate the basal transcription of protein-coding genes via interaction of the Inr with a unique Inr-binding protein, IBP39, to recruit ␣-ammanitin-resistant RNA polymerase II (17,20,22,30). This basal transcription machinery significantly differs from that of higher eukaryotic systems, in which the ␣-ammanitin-sensitive RNA polymerase II machinery exhibits considerable diversity both in the core promoter context and in the components of the promoter-recognition TFIID complex (15,24,31). Thus, the transcription efficiency of a particular type II...

Multifarious transcription of the adhesion protein ap65-1 gene in the human pathogen, Trichomonas vaginalis, is critically regulated by the coordination of two similar but opposite oriented DNA regulatory regions, MRE-1/MRE-2r and MRE-2f, both of which are binding sites for multiple Myb-like proteins. In the present study, MRE-1/MRE-2r was demonstrated to be composed of multiple overlapping promoter elements, among which the entire region is required for growth-related ap65-1 transcription, and the 5-MRE-1 antagonizes the suppressive activity of the 3-MRE-2r in iron-inducible transcription. The recombinant Myb2 protein derived from a previously identified myb2 gene was demonstrated to recognize distinct sequence contexts in MRE-2r and MRE-2f, whereas Myb2 in the nuclear lysate preferentially binds to MRE-2f to MRE-2r. Iron repletion resulted in persistent repression of the myb2 gene, and temporal activation/deactivation of Myb2 promoter entry, which was also activated by prolonged iron depletion. The hemagglutinintagged Myb2 when overexpressed during iron-depleted conditions facilitated basal and growth-related ap65-1 transcription to a level that was achieved in iron-replete cells, whereas ironinducible ap65-1 transcription was abolished with knockdown of Myb2. These findings demonstrated that Myb2 is involved in activation of growth-related and iron-inducible transcription of the ap65-1 gene, possibly through differential promoter selection in competition with other Myb proteins.Trichomonas vaginalis is a protozoan parasite that causes the most common sexually transmitted disease of nonviral origin in humans. The disease poses an imminent threat to public health as revealed by recent findings that transmission of the human immunodeficiency virus increases in patients with trichomoniasis (1). The parasite persistently inhabits the human urogenital tract without an alternating life stage outside of the host. Cytoadherence, which is crucial for T. vaginalis to establish an infection, has been shown to involve multiple surface adhesion proteins and lipophosphoglycans (2-4). The iron supply, which undergoes periodic fluctuations in the human vagina, is one of the principle determinants modulating cytoadherence of the parasite toward human vaginal epithelial cells (5, 6), possibly through transcriptional regulation of some of the adhesion protein (ap) 2 genes, especially those in the ap65 family (7, 8), which encode proteins identical to malic enzymes (9, 10). Iron has also been implicated in modulating phenotypic variation of the parasite as well as its resistance to complement lysis (11, 12). These observations underscore the importance of iron in modulating expression of parasite virulence.Gene transcription in T. vaginalis is monocistronic with only a few intron-containing genes capable of undergoing RNA splicing (13). Transcription initiation by RNA polymerase II is thus a key step in controlling expression of the protein coding genes in the parasite. Using transcription of the ap65-1 gene as a model system, we...

Background: Myb1 mediates transcription suppression of an ap65-1 gene. Results: TvCyP1 may accelerate conformational changes in Myb1 to enable its release from certain vesicles. Conclusion: TvCyP1 is essential for moving Myb1 toward the nucleus. Significance: This study elucidates a key step in nuclear translocation of Myb1 and provides tools to study the physiological role of TvCyP1.

in Trichomonas vaginalis, the TvCyP1-catalyzed conformational switches of two glycinyl-prolyl imide bonds in Myb3 were previously shown to regulate the trafficking of Myb3 from cytoplasmic membrane compartments towards the nucleus. In this study, TvCyP2 was identified as a second cyclophilin that binds to Myb3 at the same dipeptide motifs. The enzymatic proficiency of TvCyP2, but not its binding to Myb3, was aborted by a mutation of Arg 75 in the catalytic domain. TvCyP2 was localized to the endoplasmic reticulum with a weak signal that extensively extends into the cytoplasm as well as to the plasma membrane according to an immunofluorescence assay. Moreover, TvCyP2 was co-enriched with TvCyP1 and Myb3 in various membrane fractions purified by differential and gradient centrifugation. TvCyP2 was found to proficiently enzymatically regulate the distribution of TvCyP1 and Myb3 among purified membrane fractions, and to localize TvCyP1 in hydrogenosomes and on plasma membranes. Protein complexes immunoprecipitated from lysates of cells overexpressing TvCyP1 and TvCyP2 were found to share some common components, like TvCyP1, TvCyP2, TvBip, Myb3, TvHSP72, and the hydrogenosomal heat shock protein 70 (HSP70). Direct interaction between TvCyP1 and TvCyP2 was confirmed by a GST pull-down assay. Fusion of vesicles with hydrogenosomes was observed by transmission electron microscopy, whereas TvCyP1, TvCyP2, and Myb3 were each detected at the fusion junction by immunoelectron microscopy. These observations suggest that T. vaginalis may have evolved a novel protein trafficking pathway to deliver proteins among the endomembrane compartments, hydrogenosomes and plasma membranes.Trichomonas vaginalis is a p arasitic protozoan that survives only as trophozoites in the human urogenital tract 1 . Trichomoniasis has long held the position as the most common sexually transmitted disease of nonviral origin 2 . The infection often manifests mild symptoms or is asymptomatic, but it can sometimes cause adverse outcomes during pregnancy, ranging from preterm deliveries or low birth weights to abortions and stillbirths 3,4 . Trichomoniasis is also recognized as a risk factor for the elevated transmission of the human immunodeficiency virus (HIV) and papillomaviruses, and the development of progressive cervical and prostate cancers 5-8 . Trichomoniasis can be easily cured by metronidazole, a drug commonly used for gram-negative bacterial infections and luminal giardiasis and entamebiasis, but reports of drug-resistant clinical isolates have been increasing over the years 3 . The infection is often overlooked, and transmission of T. vaginalis is difficult to control 9 , rendering this neglected parasite an emerging threat to public health.Among environmental factors, iron was shown to modulate the virulence of this parasite via its effects on the transcription of myriad genes 10-12 . Accordingly, iron was shown to regulate expression levels, protein trafficking, and promoter entry of three transcription factors, Myb1, Myb2, and Myb3, which i...

Background: Iron induces the immediate nuclear influx of Myb3 in T. vaginalis. Results: Iron triggered a cAMP-mediated signaling that resulted in the phosphorylation and ubiquitination of Myb3 to accelerate its nuclear influx. Conclusion: Iron triggers signal transduction to activate a rapid nuclear influx of Myb3. Significance: This work revealed a novel role of iron in poorly studied signal transduction in the parasite.

In Trichomonas vaginalis, a TvCyP1 cyclophilin was previously demonstrated to regulate the nuclear translocation of Myb1 and Myb3, which respectively repress and activate transcription of an adhesion protein ap65-1 gene. In the present study, TvCyP1 was found to bind to Myb3 at sites spanning Gly-Pro and Gly-Pro with differential affinities. When Gly and Gly in Myb3 were both mutated, the mutant protein was restrained on outer membranes of hydrogenosomes and some cytoplasmic vesicles. In the purified Myb3 protein complex, a high molecular weight Myb3-interacting protein (Myb3IP ) and a 72-kDa heat shock protein (TvHSP72) were identified and characterized, with direct binding of Myb3 to Myb3IP and TvHSP72 confirmed in vitro. When cell lysates were fractionated by the differential and gradient centrifugations, TvCyP1 and Myb3 were always associated with membrane fractions enriched with Myb3IP and Myb1, as well as hydrogenosomes and V organelle fractions. Mutations of Gly and/or Gly resulted in membrane redistribution of Myb3 and the aberrant assembly of the Myb3 protein complex. Consistent with these findings, the involvement of TvCyP1 in membrane distribution of Myb3, and dissociation of Myb3 from TvCyP1 protein complex were demonstrated, with direct interactions between TvCyP1 and Myb3IP and that between TvCyP1 and TvHSP72, confirmed in vitro. These observations suggest that TvCyP1 directly binds to Myb3 and some of its interacting proteins to mediate serial conformational switches of Myb3 for its transition from the membrane compartments toward the nucleus.

Nuclear proteins usually contain specific peptide sequences, referred to as nuclear localization signals (NLSs), for nuclear import. These signals remain unexplored in the protozoan pathogen, Trichomonas vaginalis. The nuclear import of a Myb2 transcription factor was studied here using immunodetection of a hemagglutinin-tagged Myb2 overexpressed in the parasite. The tagged Myb2 was localized to the nucleus as punctate signals. With mutations of its polybasic sequences, 48KKQK51 and 61KR62, Myb2 was localized to the nucleus, but the signal was diffusive. When fused to a C-terminal non-nuclear protein, the Myb2 sequence spanning amino acid (aa) residues 48 to 143, which is embedded within the R2R3 DNA-binding domain (aa 40 to 156), was essential and sufficient for efficient nuclear import of a bacterial tetracycline repressor (TetR), and yet the transport efficiency was reduced with an additional fusion of a firefly luciferase to TetR, while classical NLSs from the simian virus 40 T-antigen had no function in this assay system. Myb2 nuclear import and DNA-binding activity were substantially perturbed with mutation of a conserved isoleucine (I74) in helix 2 to proline that altered secondary structure and ternary folding of the R2R3 domain. Disruption of DNA-binding activity alone by point mutation of a lysine residue, K51, preceding the structural domain had little effect on Myb2 nuclear localization, suggesting that nuclear translocation of Myb2, which requires an ordered structural domain, is independent of its DNA binding activity. These findings provide useful information for testing whether myriad Mybs in the parasite use a common module to regulate nuclear import.

Cytoadherence of Trichomonas vaginalis to human vaginal epithelial cells (hVECs) was previously shown to involve surface lipoglycans and several reputed adhesins on the parasite. Herein, we report some new observations on the host-parasite interactions of adherent versus nonadherent T. vaginalis isolates to hVECs. The binding of the TH17 adherent isolate to hVECs exhibited an initial discrete phase followed by an aggregation phase inhibited by lactose. T. vaginalis infection immediately induced surface expression of galectin-1 and -3, with extracellular amounts in the spent medium initially decreasing and then increasing thereafter over the next 60 min. Extracellular galectin-1 and -3 were detected on the parasite surface but only the TH17 adherent isolate could uptake galectin-3 via the lysosomes. Only the adherent isolate could morphologically transform from the round-up flagellate with numerous transient protrusions into a flat amoeboid form on contact with the solid surface. Cytochalasin D challenge revealed that actin organization was essential to parasite morphogenesis and cytoadherence. Real-time microscopy showed that parasite exploring and anchoring on hVECs via the axostyle may be required for initial cytoadherence. Together, the parasite cytoskeleton behaviors may collaborate with cell surface adhesion molecules for cytoadherence. The nonadherent isolate migrated faster than the adherent isolate, with motility transiently increasing in the presence of hVECs. Meanwhile, differential histone acetylation was detected between the two isolates. Also, TH17 without Mycoplasma symbiosis suggests that symbiont might not determine TH17 innate cytoadherence. Our findings regarding distinctive host-parasite interactions of the isolates may provide novel insights into T. vaginalis infection.