Immunolocalization of <i>P</i>. <i>berghei</i> PHIST proteins PBANKA_114540, PBANKA_122900, and IBIS within infected erythrocytes.

<p><b>A</b>) PBANKA_114540 co-localizes with PBANKA_122900 within erythrocyte cytoplasmic vesicles. Co-localization was assayed using rabbit polyclonal anti-PBANKA_114540 followed by goat anti-rabbit IgG conjugated with Alexa 488 (green), followed by rabbit polyclonal anti-PBANKA_122900 followed by goat anti-rabbit Alexa 595 (red). Nuclei were stained with DAPI (blue). Control experiments using secondary antibodies were negative. <b>B</b>) Co-localization of mCherry-tagged IBIS protein PBANKA_136550 with the PHIST protein PBANKA_122900 in fixed erythrocytes, by staining with rabbit polyclonal anti-PBANKA_122900 followed by goat anti-rabbit IgG conjugated with Alexa 488. Parasite nuclei were stained with Hoechst (blue). Both proteins partially co-localize inside vesicles in the erythrocyte cytoplasm.</p

Protein structure of the <i>phist</i> genes <i>PFE1600w</i> and <i>PFE1605w</i>.

A) PFE1600w and PFE1605w possess a similar protein architecture, composed of a recessed signal peptide sequence (yellow rectangle) followed by a PEXEL/HT motif (red rectangle) and a PHIST domain (green rectangle). The numbers in the schematics indicate the lengths in aa for each protein. B) Amino acid sequence alignment of the PHIST domains from PFE1600w and PFE1605w. Tryptophan residues are shaded in gray. The predicted helical segments are shown above the alignment, marked by “H”. Below the alignment, stars indicate identical aa residues, 2 dots indicate conserved substitutions and one dot indicates semi-conserved substitutions.</p

Transcript expression analysis of <i>phist</i> and <i>resa-like</i> genes during <i>P</i>. <i>falciparum</i> intraerythrocytic stages.

<p>Gene-specific transcript levels for select <i>phist</i> genes <b>A</b>) and for the <i>resa-like</i> genes <b>B</b>) were analyzed by real time PCR using cDNA prepared from intraerythrocytic synchronized asexual and gametocyte <i>P</i>. <i>falciparum</i> cultures. Note that <i>resa</i> (<i>PFA0110w</i>) and <i>resa2</i> (<i>PF11_0509</i>) are highly expressed relative to the other <i>resa-like</i> genes. Transcript expression was normalized to the expression of the control gene <i>arginyl tRNA synthetase</i> (<i>PFL0900c</i>). Hours post-synchronization indicate time in hours after adding purified schizonts to fresh blood culture. In the schematic depiction of <i>phist</i> and <i>resa-like</i> genes, yellow represents the signal sequence, and red represents the PEXEL/HT motif. The life cycle stage percentage in the population for each time point is shown in <b>C</b>). The letter “G” indicates mature gametocytes.</p

The <i>P</i>. <i>berghei phist</i> genes.

A) Protein domain architectures for P. berghei PHIST-domain encoding genes. The ruler above the architectures indicates protein lengths in aa, drawn to scale. Blue boxes represent the signal sequence; red boxes represent the PEXEL/HT motif; green boxes indicate the PHIST domain; and yellow boxes indicate repeat regions. B) Amino acid sequence alignment of the PHIST domains from PBANKA_114540 and PBANKA_122900. Tryptophan residues are shaded in gray. The predicted helical segments are shown above the alignment, marked by “H”. Below the alignment, stars indicate identical aa residues, 2 dots indicate conserved substitutions and one dot indicates semi-conserved substitutions. C) Transcript expression analysis of the phist genes during the P. berghei life cycle. Transcript levels for PBANKA_114540 and PBANKA_122900 throughout the parasite life cycle was analyzed by real-time quantitative RT-PCR using cDNA that was prepared from synchronized asexual stages, gametocytes and mosquito stage parasites. Transcript expression was normalized to the expression of the control gene hsp70. Pbama-1 was used as a stage-specific control for schizont transcription; PbCCp1 for gametocytes; and PbCSP for oocysts and sporozoites. 5 h, 12 h, 16 h, 20 h and 23 h indicate hours after injecting synchronized schizonts into the tail vein of a mouse. SC, purified schizonts; G, gametocytes; 5 d, 5-day oocysts; 8 d, 8-day oocysts; 10 d, 10-day oocysts; 15 d, 15-day oocysts; SG, salivary gland sporozoites. The composition of the asexual population with respect to life cycle stage is shown for each time point in D), expressed as percentages of the total population.</p

The <i>Plasmodium</i> PHIST and RESA-Like Protein Families of Human and Rodent Malaria Parasites

<div><p>The <i>phist</i> gene family has members identified across the <i>Plasmodium</i> genus, defined by the presence of a domain of roughly 150 amino acids having conserved aromatic residues and an all alpha-helical structure. The family is highly amplified in <i>P</i>. <i>falciparum</i>, with 65 predicted genes in the genome of the 3D7 isolate. In contrast, in the rodent malaria parasite <i>P</i>. <i>berghei</i> 3 genes are identified, one of which is an apparent pseudogene. Transcripts of the <i>P</i>. <i>berghei phist</i> genes are predominant in schizonts, whereas in <i>P</i>. <i>falciparum</i> transcript profiles span different asexual blood stages and gametocytes. We pursued targeted disruption of <i>P</i>. <i>berghei phist</i> genes in order to characterize a simplistic model for the expanded <i>phist</i> gene repertoire in <i>P</i>. <i>falciparum</i>. Unsuccessful attempts to disrupt <i>P</i>. <i>berghei PBANKA_114540</i> suggest that this <i>phist</i> gene is essential, while knockout of <i>phist PBANKA_122900</i> shows an apparent normal progression and non-essential function throughout the life cycle. Epitope-tagging of <i>P</i>. <i>falciparum</i> and <i>P</i>. <i>berghei phist</i> genes confirmed protein export to the erythrocyte cytoplasm and localization with a punctate pattern. Three <i>P</i>. <i>berghei</i> PEXEL/HT-positive exported proteins exhibit at least partial co-localization, in support of a common vesicular compartment in the cytoplasm of erythrocytes infected with rodent malaria parasites.</p></div

Dynamics of malaria infection after inoculation with erythrocytic stage parasites and sporozoites.

<p>Survival curves and daily parasitemia of <b>A)</b> Swiss Webster or <b>B)</b> C57Bl/6 mice injected intravenously with the indicated number of schizonts or mixed asexual stages of <i>P</i>. <i>berghei</i> ANKA wt or PBANKA_122900 KO, respectively. Each group contained 5 mice. The average parasitemia for all mice in each group is plotted. C) Survival curves of Swiss Webster mice injected intravenously with 1,000 sporozoites of <i>P</i>. <i>berghei</i> ANKA wt or PBANKA_122900 KO. Mice were monitored daily and the percentage surviving on each day is plotted. There were 5 mice per group and this experiment was repeated with similar results.</p

Immunolocalization of c-myc epitope-tagged PFE1605w PHIST protein within infected erythrocytes.

<p><b>A</b>) Synchronized parasites were harvested at different time points after Percoll-sorbitol purification, air-dried and fixed with ice-cold methanol. Epitope-tagged proteins were stained with anti-c-myc monoclonal antibodies that were conjugated with FITC. Nuclei were stained with DAPI (blue). The abbreviations used to describe parasite stages are as follows: 22 h, late ring stage; 30 h, early-trophozoite stage; 42 h, mid-trophozoite stage; 48 h, late trophozoite stage; and schizont stage. <b>B)</b> Co-localization was assayed using rabbit polyclonal SBP1 followed by goat anti-rabbit IgG conjugated with Alexa 594 (red). <b>C)</b> The pattern of expression of c-myc epitope-tagged PFE1605w PHIST protein is dependent on the fixation protocol. When infected erythrocytes are fixed in solution with 1% paraformaldehyde, 0.075% glutaraldehyde, then PHIST protein shows a broader punctate expression than the Maurer’s cleft marker, SPB1.</p

Transcript expression analysis of <i>phist</i> and <i>resa-like</i> genes in 4 <i>P</i>. <i>falciparum</i> isolates.

<p>The transcript levels of select <i>phist</i> and <i>resa-like</i> genes in 4 isolates of <i>P</i>. <i>falciparum</i>, NF54, Dd2, HB3 and IT4, were compared by real time PCR using cDNA prepared from late schizont stage parasites. Transcript expression was normalized to the expression of the control gene <i>arginyl tRNA synthetase</i> (<i>PFL0900c</i>). In the schematic depiction of <i>phist</i> and <i>resa-like</i> genes, yellow represents the signal sequence, and red represents the PEXEL/HT motif.</p

Viability of 3D7 stage V GIE treated with different inhibitors.

<p>3D7 Stage V GIE were treated with the inhibitors for the indicated times, then washed and their viability evaluated with the colorimetric pLDH assay either immediately (time 0) or after 72 h incubation at 37°C, as described [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004815#ppat.1004815.ref056" target="_blank">56</a>]. The data are expressed as percent of untreated controls and are the mean of two experiments in quadruplicate.</p><p>Viability of 3D7 stage V GIE treated with different inhibitors.</p

Sildenafil impairs mature GIE filterability.

<p><b>A</b>. Stage V GIE were harvested by magnetic isolation and incubated at 37°C for 30 min with 100 μM sildenafil, or 0.1% DMSO (Control). The total intracellular cAMP concentration was measured on aliquots of 6.10⁶ cells in duplicate wells. The assay was carried out five times. Error bars denote the standard error of the mean. **Highly significant differences in retention rates compared to control (DMSO) (<i>P <</i> 0.01). <b>B.</b> Graphical representation for the proportion of GIE showing a regular (light green), or deformed (dark green) shape in a population of paraformaldehyde-fixed GIE, as they flow through the microsphilters after pre-incubation at 37°C for 30 min with 100μM sildenafil, or 0.1% DMSO (Control). <b>C</b>. Differential interference contrast images of paraformaldehyde-fixed GIE, as they flow through the microsphilters. <b>D</b>. Retention rates in microsphilters of stage V GIE (light grey) and uninfected red blood cells (uRBC, dark grey) pre-incubated 30 min at 37°C with different concentrations of sildenafil. Error bars denote the standard error of the mean. Outliers are shown as open circles. *** and **Highly significant differences in retention rates compared to control (**<i>P <</i> 0.01; ***<i>P <</i> 0.001); ns: non-significant differences in retention rates compared to control. n: number of experiments.</p