Distinct features of the host-parasite interactions between nonadherent and adherent Trichomonas vaginalis isolates.

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.

Neuronal paxillin and drebrin mediate BDNF-induced force transduction and growth cone turning in a soft-tissue-like environment.

Soft tissue environments govern neuronal morphogenesis. However, the precise molecular mechanisms underlying chemotropism-directed axonal growth cone movement in extremely soft environments remain unclear. Here, we show that drebrin, a growth cone T-zone protein, modulates growth cone turning in response to brain-derived neurotrophic factor (BDNF) coated on a soft substrate. Structurally, axonal growth cones of rodent hippocampal neurons grown on 0.1 kPa hydrogels possess an expanded T zone in which drebrin is highly integrated with both F-actin and microtubules. Biochemically, we identify paxillin as interacting with drebrin in cells grown on 0.1 kPa hydrogels but not on glass coverslips. When grown on 0.1 kPa substrates, growth cones asymmetrically exposed to BDNF-bound stripes exhibit enhanced paxillin-drebrin interaction on the side facing the stripes, an activity that is PKA and AAK1 dependent but independent of Src kinase. Functionally, we show that BDNF-induced growth cone turning and force generation on soft substrates require drebrin phosphorylation and paxillin-drebrin association.

Comprehensive Workflow for Mapping Disulfide Linkages Including Free Thiols and Error Checking by On-Line UV-Induced Precolumn Reduction and Spiked Control.

Mapping highly complicated disulfide linkages and free thiols via liquid chromatography-tandem mass spectrometry (LC-MS2) is challenging because of the difficulties in optimizing sample preparation to acquire critical MS data and detecting mispairings. Herein, we report a highly efficient and comprehensive workflow using an on-line UV-induced precolumn reduction tandem mass spectrometry (UV-LC-MS2) coupled with two-stage data analysis and spiked control. UV-LC-MS2 features a gradient run of acetonitrile containing a tunable percentage of photoinitiators (acetone/alcohol) that drives the sample to the MS through a UV-flow cell and reverse phase column to separate UV-induced products for subsequent fragmentation via low energy collision-induced dissociation. This allowed the alkylated thiol-containing and UV-reduced cysteine-containing peptides to be identified by a nontargeted database search. Expected or unexpected disulfide/thiol mapping was then carried out based on the search results, and data were derived from partially reduced species by photochemical reaction. Complete assignments of native and scrambled disulfide linkages of insulin, α-lactalbumin, and bovine serum albumin (BSA) as well as the free C34-BSA were demonstrated using none or single enzyme digestion. This workflow was applied to characterize unknown disulfide/thiol patterns of the recombinant cyclophilin 1 monomer (rTvCyP1 mono) from the human pathogen Trichomonas vaginalis. α-Lactalbumin was judiciously chosen as a spiked control to minimize mispairings due to sample preparation. rTvCyP1 was determined to contain a high percentage of thiol (>80%). The rest of rTvCyP1 mono were identified to contain two disulfide/thiol patterns, of which C41-C169 linkage was confirmed to exist as C53-C181 in rTvCyP2, a homologue of rTvCyP1. This platform identifies heterogeneous protein disulfide/thiol patterns in a de-novo fashion with artifact control, opening up an opportunity to characterize crude proteins for many applications.

N-Terminal Segment of TvCyP2 Cyclophilin from Trichomonas vaginalis Is Involved in Self-Association, Membrane Interaction, and Subcellular Localization.

In Trichomonas vaginalis (T. vaginalis), cyclophilins play a vital role in dislodging Myb proteins from the membrane compartment and leading them to nuclear translocation. We previously reported that TvCyP1 cyclophilin from T. vaginalis forms a dimer and plays an essential role in moving the Myb1 transcription factor toward the nucleus. In comparison, TvCyP2 containing an extended segment at the N-terminus (N-terminal segment) formed a monomer and showed a different role in regulating protein trafficking. Four X-ray structures of TvCyP2 were determined under various conditions, all showing the N-terminal segment interacting with the active site of a neighboring TvCyP2, an unusual interaction. NMR study revealed that this particular interaction exists in solution as well and also the N-terminal segment seems to interact with the membrane. In vivo study of TvCyP2 and TvCyP2-∆N (TvCyP2 without the N-terminal segment) indicated that both proteins have different subcellular localization. Together, the structural and functional characteristics at the N-terminal segment offer valuable information for insights into the mechanism of how TvCyP2 regulates protein trafficking, which may be applied in drug development to prevent pathogenesis and disease progression in T. vaginalis infection.

Endomembrane Protein Trafficking Regulated by a TvCyP2 Cyclophilin in the Protozoan Parasite, Trichomonas vaginalis.

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 Arg75 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.

Membrane localization of a Myb3 transcription factor regulated by a TvCyP1 cyclophilin in the parasitic protozoan Trichomonas vaginalis.

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 54 Gly-Pro55 and 72 Gly-Pro73 with differential affinities. When Gly54 and Gly72 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 (Myb3IPhmw ) and a 72-kDa heat shock protein (TvHSP72) were identified and characterized, with direct binding of Myb3 to Myb3IPhmw 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 Myb3IPhmw and Myb1, as well as hydrogenosomes and VMyb organelle fractions. Mutations of Gly54 and/or Gly72 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 Myb3IPhmw 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.

Signal transduction triggered by iron to induce the nuclear importation of a Myb3 transcription factor in the parasitic protozoan Trichomonas vaginalis.

Iron was previously shown to induce rapid nuclear translocation of a Myb3 transcription factor in the protozoan parasite, Trichomonas vaginalis. In the present study, iron was found to induce a transient increase in cellular cAMP, followed by the nuclear influx of Myb3, whereas the latter was also induced by 8-bromo-cyclic AMP. Iron-inducible cAMP production and nuclear influx of Myb3 were inhibited by suramin and SQ22536, respective inhibitors of the Gα subunit of heterotrimeric G proteins and adenylyl cyclases. In contrast, the nuclear influx of Myb3 induced by iron or 8-bromo-cAMP was delayed or inhibited, respectively, by H89, the inhibitor of protein kinase A. Using liquid chromatography-coupled tandem mass spectrometry, Thr(156) and Lys(143) in Myb3 were found to be phosphorylated and ubiquitinated, respectively. These modifications were induced by iron and inhibited by H89, as shown by immunoprecipitation-coupled Western blotting. Iron-inducible ubiquitination and nuclear influx were aborted in T156A and K143R, but T156D was constitutively ubiquitinated and persistently localized to the nucleus. Myb3 was phosphorylated in vitro by the catalytic subunit of a T. vaginalis protein kinase A, TvPKAc. A transient interaction between TvPKAc and Myb3 and the phosphorylation of both proteins were induced in the parasite shortly after iron or 8-bromo-cAMP treatment. Together, these observations suggest that iron may induce production of cAMP and activation of TvPKAc, which then induces the phosphorylation of Myb3 and subsequent ubiquitination for accelerated nuclear influx. It is conceivable that iron probably exerts a much broader impact on the physiology of the parasite than previously thought to encounter environmental changes.

Regulation of nuclear translocation of the Myb1 transcription factor by TvCyclophilin 1 in the protozoan parasite Trichomonas vaginalis.

In Trichomonas vaginalis, a Myb1 protein was previously demonstrated to repress transcription of an iron-inducible ap65-1 gene. In this study, a human cyclophilin A homologue, TvCyclophilin 1 (TvCyP1), was identified as a Myb1-binding protein using a bacterial two-hybrid library screening system. The recombinant TvCyP1 exhibited typical peptidyl-prolyl isomerase activity with kcat/Km of ∼7.1 µm(-1) s(-1). In a pulldown assay, the His-tagged Myb1 interacted with a GST-TvCyP1 fusion protein, which had an enzymatic proficiency half that of recombinant TvCyP1. Both the enzymatic proficiency of GST-TvCyP1 and its binding to His-Myb1 were eliminated by mutation of Arg(63) in the catalytic motif or inhibited by cyclosporin A. TvCyP1 was primarily localized to the hydrogenosomes by immunofluorescence assay, but it was also co-purified with Myb1 in certain vesicle fractions from differential and gradient centrifugations. Transgenic cells overexpressing HA-TvCyP1 had a higher level of nuclear Myb1 but a much lower level of Myb1 associated with the vesicles than control and those overexpressing HA-TvCyP1(R63A). Myb1 was detected at a much higher level in the HA-TvCyP1 protein complex than in the HA-TvCyP1(R63A) protein complex immunoprecipitated from P15 and P100, but not S100, fractions of postnuclear lysates. A TvCyP1-binding motif, (105)YGPKWNK(111), was identified in Myb1 in which Gly(106) and Pro(107) were essential for its binding to TvCyP1. Mutation of Gly(106) and Pro(107), respectively, in HA-Myb1 resulted in cytoplasmic retention and elevated nuclear translocation of the overexpressed protein. These results suggest that TvCyP1 may induce the release of Myb1 that is restrained to certain cytoplasmic vesicles prior to its nuclear translocation.

A highly organized structure mediating nuclear localization of a Myb2 transcription factor in the protozoan parasite Trichomonas vaginalis.

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.

Activation of multifarious transcription of an adhesion protein ap65-1 gene by a novel Myb2 protein in the protozoan parasite Trichomonas vaginalis.

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.

Multifarious transcriptional regulation of adhesion protein gene ap65-1 by a novel Myb1 protein in the protozoan parasite Trichomonas vaginalis.

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.