Transcriptional Regulation of an Iron-Inducible Gene by Differential and Alternate Promoter Entries of Multiple Myb Proteins in the Protozoan Parasite Trichomonas vaginalis

Abstract

Iron-inducible transcription of a malic enzyme gene (also reputed to be ap65-1) in Trichomonas vaginalis was previously shown to involve a Myb1 repressor and a Myb2 activator, each of which may preferentially select two closely spaced promoter sites, MRE-1/MRE-2r, which comprises overlapping promoter elements, and MRE-2f. In the present study, an iron-inducible ϳ32-kDa Myb3 nuclear protein was demonstrated to bind only the MRE-1 element. Changes in the iron supply, which produced antagonistic effects on the levels of Myb2 and Myb3 expression, also resulted in temporal and alternate entries of Myb2 and Myb3 into the ap65-1 promoter. Repression or activation of basal and iron-inducible ap65-1 transcription was detected in transfected cells when Myb3 was, respectively, substantially knocked down or overexpressed. In the latter case, increased Myb3 promoter entry was detected with concomitant decrease in Myb2 promoter entry under specific conditions, while Myb3 promoter entry was inhibited under all test conditions in cells overexpressing Myb2. In contrast, concomitant promoter entries by Myb2 and Myb3 diminished in cells overexpressing Myb1, except that Myb3 promoter entry was slightly affected under prolonged iron depletion. Together, these results suggest that Myb2 and Myb3 may coactivate basal and iron-inducible ap65-1 transcription against Myb1 through conditional and competitive promoter entries.

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

“…1A), which drives protein expression to a higher level than a previous version for better detection (18). HA-Myb3 was mainly localized in nuclei, with much weaker punctate signals also observed in the cytoplasm of all transfected cells examined, but not the control, according to an IFA using a mouse anti-HA antibody (Fig.…”

“…To generate a plasmid with a higher HA-tagged Myb3 (HA-Myb3) expression level than that conferred by pAPm(MRE-1)-ha-myb3, the sequence encompassing the ap65-2.1 promoter was amplified from pAP65-2.1-ha-myb2/TUBneo (30) by a PCR using the primer pair pAP65-2.1-sac2-5= and ap65-2.1-hind3-3= (see Table S1 in the supplemental material). The amplified DNA was restricted by SacII/HindIII and subcloned into a vector backbone from SacII/NsiIdigested pAPm(MRE-1)-ha-myb3 (18), along with the HindIII/NsiI fragment from pAPm(MRE-1)-ha-myb3, to generate pAP65-2.1-ha-myb3 (Fig. 1A).…”

“…Myb1, Myb2, Myb3, and CyPA1 were each detected using mouse antiMyb1 (1,000ϫ), rabbit anti-Myb2 (3,000ϫ), rabbit anti-Myb3 (3,000ϫ), and mouse anti-CyPA1 (5,000ϫ), respectively. The signal intensity of each protein on a blot was measured and quantified as previously described (18).…”

“…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.…”

Epitopes of the Highly Immunogenic Trichomonas vaginalis α-Actinin Are Serodiagnostic Targets for Both Women and Men

“…Reports have shown the expression of numerous virulence factors that permit this ancient protist to survive in the changing host environment of the female urogenital region (3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18). Iron is an important modulator of upregulation and downregulation of the expression of virulence genes (8,(19)(20)(21)(22)(23)(24)(25). Key steps for host colonization result from overcoming the urogenital mucous layer (18) in preparation for specific cytoadherence to vaginal epithelial cells (7,9,11,22,24).…”