Activation of Multifarious Transcription of an Adhesion Protein ap65-1 Gene by a Novel Myb2 Protein in the Protozoan Parasite Trichomonas vaginalis

Abstract

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...

Iron-Inducible Nuclear Translocation of a Myb3 Transcription Factor in the Protozoan Parasite Trichomonas vaginalis

“…To date, multiple parasitic factors that contribute to cytoadherence, including some carbohydrate moieties and several reputed adhesion proteins on plasma membranes, have been identified (1-6, 16, 26). Serial studies on inducible transcription of the adhesion protein gene, ap65-1, revealed that its transcription is critically coregulated by three Myb proteins, Myb1, Myb2, and Myb3, through competitive entries of multiple Myb recognition elements in the distal promoter region in a space-and time-dependent manner (18,(28)(29)(30)38), suggesting that these Myb proteins are differentially regulated in response to various stimuli from host environments. Myb proteins are ubiquitous in eukaryotes that share a conserved DNA-binding domain (DBD) composed of one to three repeat sequences termed R1, R2R3, and R1R2R3.…”

A Highly Organized Structure Mediating Nuclear Localization of a Myb2 Transcription Factor in the Protozoan Parasite Trichomonas vaginalis

“…Sequences of the oligonucleotides used in the present study are listed in Table S1 in the supplemental material or were reported in previous studies (15,35). The plasmid, pAP65-2.1-ha-myb2/TUBneo, which can overexpress Myb2 with a single copy of an N-terminal hemagglutinin (HA) tag was obtained from a previous study (35).…”

“…Sequences of the oligonucleotides used in the present study are listed in Table S1 in the supplemental material or were reported in previous studies (15,35). The plasmid, pAP65-2.1-ha-myb2/TUBneo, which can overexpress Myb2 with a single copy of an N-terminal hemagglutinin (HA) tag was obtained from a previous study (35). To increase the detection sensitivity of HA-Myb2 in T. vaginalis, a modified vector, pAP65-2.1-4ha-myb2/ TUBneo, which can overexpress Myb2 with four copies of the HA tag, was constructed by a two-step PCR as described below.…”

“…Two transcription activators, Myb2 and Myb3, and a Myb1 repressor were demonstrated to coregulate inducible transcription of the ap65-1 gene (15,34,35,44), which encodes a 65-kDa malic enzyme that may also serve as an adhesion protein under iron-replete conditions (21,31). The parasite genome encodes a super family of Myb proteins comprising Ͼ400 members (3), most of which share a conserved R2R3 DNA-binding domain (DBD), which comprises six ␣-helices spanning ϳ100 amino acid (aa) residues (19,28,33), and highly variable N and C termini.…”

“…To date, only a few nuclear proteins have been studied in this parasite (15,24,27,34,35,38). The molecular masses of these nuclear proteins are Ͻ40 kDa, the sizes of which may allow free diffusion across the nuclear pores (42).…”