Evidence for the Nucleo-Apical Shuttling of a Beta-Catenin Like Plasmodium falciparum Armadillo Repeat Containing Protein.

Eukaryotic Armadillo (ARM) repeat proteins are multifaceted with prominent roles in cell-cell adhesion, cytoskeletal regulation and intracellular signaling among many others. One such ARM repeat containing protein, ARM Repeats Only (ARO), has recently been demonstrated in both Toxoplasma (TgARO) and Plasmodium (PfARO) parasites to be targeted to the rhoptries during the late asexual stages. TgARO has been implicated to play an important role in rhoptry positioning i.e. directing the rhoptry towards the apical end of the parasite. Here, we report for the first time that PfARO exhibits a DNA binding property and a dynamic sub-cellular localization between the nucleus (early schizont) and rhoptry (late schizont) during the different stages of the asexual blood-stage life cycle. PfARO possesses a putative nuclear export signal (NES) and the nucleo-apical shuttling was sensitive to Leptomycin B (LMB) suggesting that the nuclear export was mediated by CRM1. Importantly, PfARO specifically bound an A-T rich DNA sequence of the P. falciparum Gyrase A (PfgyrA) gene, suggesting that the DNA binding specificity of PfARO is likely due to the AT-richness of the probe. This is a novel functional characteristic that has not been reported previously for any P. falciparum ARM containing protein and suggests a putative role for PfARO in gene regulation. This study describes for the first time a conserved P. falciparum ARM repeat protein with a high degree of functional versatility

Native PfARO exhibits nucleo-apical shuttling.

<p><b>A.</b> Stage specific expression analysis of native PfARO during the intra-erythrocytic asexual 48 hour cycle by immunoblotting using specific PfARO antibodies. PfARO expression is initiated at early schizont stages (30–32 hours post infection) and peaks at late schizont stages (44–48 hpi). Expression of the constitutive parasite protein, PfActin was analyzed as a control. <b>B.</b> Confocal immunofluorescence imaging using PfARO specific antibodies detected predominant expression of native PfARO in the nucleus during the early schizont stage parasites, which decreased with the intra-erythrocytic growth of the parasite. At later stages, native PfARO was observed to be predominantly outside the nucleus (Scale bar, 2 μm). <b>C.</b> Stage specific nuclear fractionation followed by immunoblot analysis was consistent with the imaging results as It native PfARO was detected in the nuclear fraction in early schizont stage (32 hpi) while it is mostly non-nuclear or cytosolic at the late schizont stages (44 hpi). As controls, Histone H3 was found exclusively in the nuclear fraction; Aldolase was found in the cytosolic fraction as was the apically localized rhoptry protein, PfRH2. The ER resident protein marker Bip was also found to be in the cytosolic fraction. <b>D</b>. Native PfARO remained intact and protected from Proteinase K (PK) degradation in digitonin (D) permeabilized early schizont stage parasites (32 hpi) but was susceptible to PK action at the late stages (44 hpi). Other organellar proteins like the nuclear protein Histone H3 and rhoptry protein PfRH2 also remained protected from PK action in the permeabilized parasite infected erythrocytes, while the cytosolic protein GAPDH was lost on permeabilization.</p

Expression and characterization of Recombinant PfARO (rPfARO).

<p><b>A.</b> Schematic representation of the domain structure of <i>P</i>. <i>falciparum</i> ARO (PfARO) showing the N-terminal acylation region, ARM repeat domains and putative nuclear export signal. <b>B.</b> SDS-PAGE of purified recombinant rPfARO run under reducing (R) and non-reducing conditions (NR). PfARO under non-reducing condition shows a prominent band migrating at a dimeric position in addition to the monomeric form. Both monomeric and dimeric forms were recognized by anti-His antibodies in immunoblot analysis (right panel). <b>C.</b> Immunoblot analysis of recombinant PfARO using anti-His antibodies in the presence of increasing concentrations of the reducing agent, Dithiothreitol (DTT). The dimeric form of rPfARO diminished progressively with the increasing DTT concentrations suggesting a disulphide mediated homodimerization.</p

Expression of the native PfARO parasite protein and analysis of its solubility in the merozoite.

<p><b>A.</b> PfARO antibodies specifically detected native PfARO at the expected molecular mass (~32 kDa) in detergent based lysates of schizont stage parasites. No cross-reactivity was observed with any other member of the ARO family of protein or with human erythrocyte proteins from the lysate of uninfected erythrocytes/red blood cells (RBCs). Immunoblot analysis under identical experimental conditions using pre-immune (PI) sera also did not exhibit any cross-reactivity by detecting any other parasite proteins. <b>B.</b> Under non-reducing conditions, native PfARO was also detected as a dimer(~60KDa) in addition to the monomeric protein (~32 KDa). <b>C.</b> Native PfARO was specifically detected by immunoblot analysis in a detergent based lysate of the merozoite stage parasites and culture supernatant. Native EBA-175, a key invasion protein ligand, was detected as a control in both merozoite lysate and culture supernatant. <b>D.</b> PfARO mouse antibodies (immune sera, IM) specifically immunoprecipitated native PfARO from detergent based lysates of late schizont stage parasites. Pre-immune sera failed to immunoprecipitate native PfARO. The immunoprecipitated native PfARO protein were detected in immunoblots using the same PfARO mouse sera that detected the mouse IgG heavy chain (denoted in the Fig by an asterisk). Input represents the lysate sample used for immunoprecipitation. <b>E.</b> PfARO was found to be present in the supernatants obtained from the <i>P</i>. <i>falciparum</i> parasites subjected to hypotonic lysis, sodium carbonate extraction, Triton X-100 extraction as well as the insoluble fraction. GPI anchored parasite protein, CyRPA, was found predominantly in the Triton X-100 detergent extracted and insoluble fractions suggesting that it was tightly membrane associated, while the cytosolic native PfGAPDH protein was as expected found to be in the supernatant following hypotonic lysis.</p

Leptomycin B (LMB) abrogates PfARO export out of the nucleus.

<p><b>A.</b> Localization of PfARO in the parasite at late stages is affected in presence of the nucleo-cytoplasmic transport inhibitor LMB. In untreated parasites PfARO is apically located at late schizont stages. Upon treatment with LMB, PfARO remains within the nucleus as shown by confocal immunofluorescence analysis. DAPI was used for staining the nucleus and it is clear that the PfARO signal colocalizes with the DAPI staining. <b>B.</b> Under the same experimental conditions, the localization of the rhoptry protein PfRH2 remains unaffected. (Scale bar, 2 μm). <b>C</b>. Sub-cellular fractionation followed by immunoblot analysis further confirmed the enrichment of PfARO in the nuclear fraction in presence of LMB with respect to untreated parasites. Histone H3 and aldolase are used as nuclear and cytosolic markers, respectively.</p

Colocalization of native PfARO with nuclear marker at early schizont stage and rhoptry markers at late schizont stage as demonstrated by confocal immunofluorescence imaging.

<p><b>A.</b> PfARO co-localizes with the nuclear marker protein, Histone H3 at the early schizont stages. <b>B.</b> PfARO is apically located at the late schizont stages as demonstrated by its co-localization with rhoptry neck marker PfRH2. <b>C.</b> PfARO is apically located at the late schizont stages as demonstrated by its co-localization with rhoptry bulb marker PfRH5. <b>D.</b> PfARO co-localizes with PfRH2 at the apical end of the parasites in free merozoites. <b>E.</b> PfARO does not co-localize with the ER resident protein Bip either at early or late schizont stages of the parasite. DAPI was used for staining the nucleus. (Scale bar, 2 μm).</p

PfARO exhibits DNA binding activity.

<p><b>A.</b> Purified full length recombinant PfARO binds to double stranded radiolabeled DNA as demonstrated by the Electrophoretic Mobility Shift assay (EMSA). The retardation of the DNA-protein complex with respect to the free probe is clearly shown. A super-shift in the mobility of the DNA-protein complex was specifically observed in the presence of the PfARO antibodies while no mobility shift was detected in presence of pre-immune sera. <b>B.</b> Recombinant PfRH2₄₀ protein does not bind to double stranded radiolabeled DNA probe under the same experimental conditions in which rPfARO exhibits DNA binding activity. <b>C.</b> A competition assay was performed using cold AT-rich or GC-rich competitor DNA probes at varying concentrations (1X, 2X). The cold AT rich DNA abrogated the DNA binding of PfARO while the presence of the GC rich cold DNA had no effect on the DNA binding activity of PfARO. <b>D.</b> Native PfARO present in the nuclear lysate specifically bound the double stranded radiolabelled DNA as demonstrated by the super-shifted complex only in the presence of PfARO specific antibodies. The PfARO specific antibodies did not exhibit any affinity for the radiolabelled probe. No super-shift was observed with the control pre-immune sera.</p

PfARO exhibits structural conservation with other ARM repeat containing proteins.

<p><b>A.</b> Three-dimensional model of rPfARO predicted through homology modeling using Phyre2 software and visualized by Jsmol. The putative nuclear export signal (NES) is depicted in green. Model dimensions (Å): X:42.005 Y:58.467 Z:60.106. <b>B.</b> Circular dichroism analysis of recombinant rPfARO showed a helical composition consistent with the presence of tandem ARM repeats. The CD spectra of a known helical protein, BSA was run as a control.</p