Parasites and mosquitoes

The P. falciparum NF54 isolate [26] was cultivated in vitro using RPMI 1640 medium supplemented with 10% heat-inactivated human serum as described [27]. Cultures were synchronized by the isolation of schizonts using a Percoll-sorbitol gradient, and placed back into culture [28]. To collect parasites throughout an intraerythrocytic developmental cycle, parasites were harvested at different time points after erythrocyte invasion, pelleted by centrifugation and lysed with 0.1% saponin in PBS. Thin smears of blood on glass slides were made at each time point and stained with Giemsa reagent for microscopy. Gametocytes were prepared by extended in vitro culturing of asexual stage parasites, and analyzed when the cultures consisted largely of stage IV and V parasites. The isolates Dd2, HB3 and IT4 [29–31] were cultivated as described above.

The gametocyte-producing reference clone, cl15cy1 (HP), of the ANKA strain of P. berghei was maintained and gametocytes produced as described [32] and the non-gametocyte-producing clone (HPE) of the ANKA strain was also used in some experiments [33]. To collect parasites from different stages of the intraerythrocytic developmental cycle, synchronized blood stage infections of P. berghei HPE clone were performed in vivo, in Swiss Webster mice, as described [34]. Thin smears of blood on glass slides were made at each time point and stained with Giemsa reagent for microscopy. Anopheles stephensi mosquitoes were reared as described [34]. P. berghei-infected mosquitoes were maintained at 19°C. Mosquito stages were obtained after feeding 3 to 5 day-old An. stephensi mosquitoes on Swiss Webster mice that were infected with either P. berghei ANKA HP or phist gene knockout clonal lines. Oocyst samples for RT-PCR consisted of infected midguts that were removed 5, 10 and 15 days post infection. To determine parasite development in the mosquito, oocysts were counted from midguts removed at day 10 post infection; midgut sporozoites were counted on day 14 post infection; and salivary gland sporozoites were quantified from dissected salivary glands on day 17–21 post infection. Live sporozoites for in vitro and in vivo assays were isolated from day 21 salivary glands as described [32].

Animals

All experiments using animals were approved by the Weill Cornell Medical College Institutional Animal Care and Use Committee (WCMC IACUC) under the Animal Protocol number 0704-613-A, or by the New York University School of Medicine Institutional Animal Care and Use Committee (NYU IACUC) under the Animal Protocol number 080413–02, and were performed in accordance with the applicable guidelines and regulations. Animals were housed in the Weill Cornell Medical College animal facility or in the New York University School of Medicine animal facility, where they received daily care and husbandry.

Six week old female Swiss Webster mice, female C57Bl/6 mice and male Wistar Kyoto rats were obtained from Taconic Biosciences, Inc. For P. berghei infection, mice and rats were injected intraperitoneally or by mosquito bite inoculations as described [32]. To generate phist gene knockout parasites, P. berghei ANKA HP for transfection was collected from Wistar rats that harbored infections between 0.5 to 3% parasitemia. Parasitemia was monitored daily by tail vein blood smears. For collection of infected blood, animals were terminally exsanguinated by cardiac puncture, followed by cervical dislocation. Animals were euthanized once experimental data was collected or if clinical symptoms of cerebral malaria were observed. Twelve week old New Zealand White rabbits (Charles River Laboratories International, Inc.) were used for production of immune sera.

RNA isolation and transcript expression analysis by real-time quantitative RT-PCR

RNA was isolated using the Trizol-based method (Invitrogen) according to the manufacturer’s instructions, treated with DNase I (Invitrogen), and purified on RNeasy MinElute Cleanup columns (Qiagen). RNA was reverse-transcribed using Superscript II that was primed with random hexanucleotides (Invitrogen). Real-time quantitative RT-PCR was performed using an ABI Prism 7900HT sequence detector (Applied Biosystems). Reactions were prepared in 20 μL volumes using a SYBR Green PCR master mix (Applied Biosystems) and 1 μM gene-specific primers. The absence of genomic DNA contamination was confirmed by PCR amplification on sham-treated RNA samples that lacked reverse transcriptase. The specificity of amplification for each PCR product was confirmed by dissociation curve analysis. The efficiency of amplification was verified by using different concentrations of genomic DNA as templates in order to calculate the median cycle threshold (CT) value for each primer pair. All primer pairs included in this study displayed the same median CT value. Relative quantification of cDNA was performed using the standard curve method (User Bulletin 2, ABI, http://www.appliedbiosystems.com) to determine the efficiency of the target and reference amplification and to quantify cDNA in each sample. The normalized expression for each gene was determined as the ratio: relative amount of target gene cDNA/ relative amount of control gene cDNA. Triplicate PCR reactions were analyzed for each sample. Transcript expression of P. falciparum genes was normalized to the expression of the control gene, arginyltRNA synthetase (PFL0900c), while the transcript expression of P. berghei genes was normalized to the expression of the control gene, Pbhsp70 (PBANKA_091440). Gene-specific primers were empirically designed for select P. falciparum PHIST domain-containing genes and the P. berghei genes, PBANKA_114540 and PBANKA_122900 (see S1 Table for primer sequences).

Epitope-tagged constructs to fuse 3 c-myc epitopes at the C-terminus of phist genes

The sequences of all PCR primers used to make constructs in this study are included in S2 Table. Epitope-tagged constructs for episomal expression were designed to fuse DNA encoding 3 c-myc epitopes at the C-terminus of the P. falciparum genes, PFE1600w and PFE1605w. The coding sequences corresponding to both genes were amplified by PCR from genomic DNA template of the P. falciparum NF54 isolate using the following primer pairs: PFE1600wSXhoI plus PFE1600wASBglII; and PFE1605wSXhoI plus PFE1605wASBglII. The PCR products were digested with XhoI and BglII and cloned into the expression vector, pHL-dhfr-3myc, which is a derivative of the pHLH1 vector [35]. pHL-dhfr-3myc contains 3 consecutive c-myc epitopes within an expression cassette that is driven by the P. falciparum 5′ hrp3 promoter and the 3′ hrp2 terminator to ensure a high expression level of the phist genes; and an hDHFR expression cassette for selection of transformed parasites with pyrimethamine. Expression vectors were introduced into the P. falciparum isolate NF54 via the erythrocyte-loading method [36]. Stable transformant parasites were selected by culturing in media containing 40 ng/ml pyrimethamine. Episomal gene expression was detected by reverse transcription and real-time quantitative PCR using specific primers PFE1600wS2 plus PFE1600wAS2; PFE1600wS2 plus PFE1600wmycAS3c; PFE1605wS2 plus PFE1605wAS2 and PFE1605whrpS3c plus PFE1605wAS2, following the protocol described in the above transcript expression analysis section. As a control for expression and localization experiments we used the pHL-dhfr-luciferase line (a gift from Kirk Deitsch), which expresses the luciferase protein and is a derivative of the pHLH1 vector [35]. To ensure that parasites were expressing the correct protein, plasmid was rescued by transforming chemically competent Escherichia coli with 100 ng of purified genomic DNA from selected transformant parasites, and plasmid nucleotide sequence was determined.

Two independent lines of transgenic parasites were established for each construct. In the transgenic populations the average copy number per parasite of the introduced chimeric PFE1600w and PFE1605w genes was determined, by quantitative real time PCR, to be approximately 3 to 4 copies (S3B Fig). Transformed parasite lines which had been in culture for more than 100 generations prior to transgene quantification (termed Line 1 in S3B Fig), exhibited variation in the average number of gene copies between generations, while parasite populations that had been kept in culture for no longer than 30 generations (termed Line 2 in S3B Fig) displayed homogeneous copy number from one generation to the next (S3B Fig). This might be due to imperfect plasmid duplication and segregation during schizogony, such that the population becomes increasingly heterogeneous as it is propagated for many generations. Transcript levels of endogenous and episomal genes reached the maximum level of expression at similar intraerythrocytic stages as determined by quantitative real time RT-PCR, suggesting that the expression vector hrp3 promoter activity, at the late ring stage, drove transgene expression coincident with the peak of expression of endogenous PFE1600w and PFE1605w (S3C Fig). However, expression of episomal PFE1600w and PFE1605w are 10- and 22-fold higher, respectively, than the cognate endogenous genes. Western blot analysis of the epitope-tagged proteins using anti-c-myc antibodies showed expression of proteins having the expected molecular weights, with no additional bands that might suggest cross-reactivity or protein processing (S3D Fig). Phenotypic analysis of parasites harboring the PFE1600w-c-myc or PFE1605w-c-myc expression vectors exhibited normal growth in comparison to wild type (wt) NF54, indicating that over-expression of these PHIST proteins are not toxic to the parasite (data not shown).

Generation of transgenic P. berghei ANKA parasites expressing mCherry-tagged PBANKA_136550

Transfection of P. berghei parasites, selection and cloning of the transgenic parasite line expressing mCherry-tagged PBANKA_136550 (IBIS1) was performed as described [37]. To generate the targeting vector, the smac targeting region within plasmid pL1419 [38] was replaced by the targeting region of PBANKA_136550. This region was amplified with the primers 6288 and 6289 (S2 Table). Transfection of parasites with this construct results in tagging of the endogenous gene with mCherry by a single cross-over integration of the construct. Correct integration at the chromosome 13 locus was determined by Southern blot analysis of digested genomic DNA or chromosomes separated by pulse-field gel (PFG) electrophoresis and hybridization with a probe corresponding to the P. berghei 3’UTR of dhfr/ts.

Antibodies

For immunofluorescence microscopy the following antibodies were used: anti-c-myc monoclonal antibody FITC conjugate (Sigma-Aldrich F2047); rabbit anti-SBP1 rabbit polyclonal antibody directed against the N-terminus of SBP1 (a gift from Dr. Brian Cooke; [39]); anti-TER-119-FITC labeled antibody (eBioscience); goat anti-mouse Alexa 488-conjugate IgG (Molecular Probes); goat anti-rabbit Alexa 488-conjugate IgG (Molecular Probes); and goat anti-rabbit Alexa 594-conjugate IgG (Molecular Probes).

For the generation of anti-P. berghei PHIST polyclonal antibodies, 30-mer peptides corresponding to the repeat region of PBANKA_114540 (CSEKKSEKKSEKKNEENSEKKSKKKSEKKN) and PBANKA_122900 (CPLKPEQEENVDPLKPEQEENIKPLKPEQK) were synthesized by the Proteomics Resource Center of the Rockefeller University with an Applied Biosystems Model 430A instrument using FMOC based chemistry. Peptides were conjugated to mariculture keyhole limpet hemocyanin (mcKLH) using the kit Imject Maleimide Activated mcKLH (Pierce 77611) according to the manufacturer’s instructions. Twelve weeks old New Zealand White rabbits (Charles River) were immunized with 100 μg of conjugated peptide in Freund’s Complete Adjuvant (Sigma-Aldrich), followed by 3 boosts with 50 μg of conjugated peptide in Freund’s Incomplete Adjuvant (Sigma-Aldrich). One rabbit was immunized with each peptide. Peptides were covalently immobilized in a beaded agarose support using the SulfoLink kit (Pierce 44895) and the resulting columns were used to affinity purify antibodies specific to PBANKA_114540 and PBANKA_122900.

For Western blot analyses, anti-c-myc monoclonal antibody (Sigma-Aldrich M4439); anti-mouse IgG peroxidase conjugated (Sigma-Aldrich A9044) and anti-rabbit IgG peroxidase conjugate (Sigma-Aldrich A0545) secondary antibodies were used.

Indirect immunofluorescence assay and immunoelectron microscopy

Parasite culture or mouse tail vein blood was spotted within the wells of teflon-coated microscope slides (Electron Microscopy Sciences, PA), air dried and fixed for 10 min in cold methanol. For blocking of nonspecific binding, the fixed cells were incubated in PBS containing 2% bovine serum albumin and 10% normal goat serum for 1 h at room temperature. The preparations were then incubated for 1 h at room temperature with the primary antibody at the following concentrations: 5 μg/ml anti-c-myc monoclonal antibody FITC conjugate; 1:500 rabbit anti-SBP1 polyclonal antibody; 2 μg/ml affinity purified rabbit anti-PBANKA_114540; and 2 μg/ml affinity purified rabbit anti-PBANKA_122900. Slides were washed in PBS and incubated for 1 h at room temperature with appropriate secondary antibody at the following concentrations: 1 μg/ml goat anti-mouse Alexa 488-conjugate IgG; 1 μg/ml goat anti-rabbit Alexa 488-conjugate IgG; 1 μg/ml anti-rabbit Alexa 594-conjugated IgG. Nuclei were stained with DAPI. Samples were analyzed in an Olympus BX-51 fluorescence microscope with an Optronics digital imaging system.

For analysis of mCherry expression of the transgenic lines, live parasites were examined by microscopy using a Leica DMR fluorescent microscope with standard FITC and Texas Red filters. Parasites nuclei were labeled by staining with 2 μmol/L Hoechst-33258 and erythrocyte surface membranes were stained with the 1:200 anti-mouse TER-119-FITC labeled antibody at room temperature for 30 min and washed with 500 μL of RPMI-1640 medium.

For confocal microscopy, P. falciparum infected erythrocytes were washed twice in PBS, fixed in 4% paraformaldehyde, 0.075% glutaraldehyde (Electron Microscopy Sciences, PA) in PBS for 20 min at room temperature, washed 3 times in PBS and spread on poly-L lysine-coated glass slides. Slides were washed twice in PBS and incubated in 0.15% glycine in PBS for 10 min at room temperature. After 3 washes in PBS, cells were permeabilized for 10 min in 0.1% Triton X-100 in PBS, and washed twice in PBS. Samples were blocked and incubated with antibodies as described above, and analyzed in a Zeiss Exciter LSM500 confocal microscope system. All indirect immunofluorescence experiments included secondary antibodies alone controls.

Western blot analysis

Infected erythrocytes were lysed in SDS sample buffer and proteins separated by SDS-PAGE and transferred to Hybond P membrane (Amersham Biosciences) according to the manufacturer’s instructions. The membranes were blocked for nonspecific binding by incubation in Tris-buffered saline (TBS) containing 5% skim milk followed by incubation for 1 h at room temperature with 2 μg/ml anti-c-myc monoclonal antibody, or 1:1,000 anti-Pf39 polyclonal antibodies. After washing in TBS containing 0.1% Triton X-100, the membranes were incubated for 1 h at room temperature with 1:80,000 anti-mouse IgG peroxidase conjugated (Sigma-Aldrich A9044).

Targeted disruption of P. berghei phist genes

The sequences of all PCR primers used in this study are included in S2 Table. PBANKA_122900 knockout (KO) parasites were generated by double homologous recombination using the targeting vector b3D.DT^H.^D (available from MR4; http://www.malaria.mr4.org) and P. berghei ANKA erythrocytic stages. Two fragments of 732 and 626 bp (shaded in gray in S5A Fig) were amplified by PCR from P. berghei ANKA genomic DNA using the following specific primer pairs: PB848ko5SKpnI plus PB848ko5ASHindIII; and PB848ko3SEcoRI plus PB848ko3ASBamHI. To derive the disruption plasmid these PCR fragments were sequentially cloned into the b3D.DT^H.^D vector using the KpnI/HindIII sites, followed by insertion into the EcoRI/BamHI sites. The disruption vector was then digested with KpnI and BamHI to release the fragment for transfection. P. berghei ANKA schizonts were collected from 2 Wistar rats and electroporated with 10 μg of DNA using the Amaxa Nucleofector (program U33) as described [37]. Selection and cloning by limiting dilution were performed in mice as described [40]. Integration of the transfected DNA at the correct location was verified by both diagnostic PCR and Southern blot analysis. PCR was performed with 100 ng of genomic DNA from wt or PBANKA_122900 KO parasites for 35 cycles and annealing temperature of 50°C using the primers a; b (identical to PB848ko5ASHindIII); b’; c (identical to PB848ko3SEcoRI); c’; and d (locations of primers shown in S5A Fig). Southern blot analysis was performed with 10 μg of genomic DNA from wt or PBANKA_122900 KO parasites, digested with ClaI and EcoRI. A 732 bp hybridization probe was amplified with the primers PB848ko5SKpnI and PB848ko5ASHindIII. The probe was labeled with digoxigenin and hybridization was performed at 48°C using DIG High Prime DNA Labeling and Detection Starter Kit II (Roche Diagnostics), according to the manufacturer’s instructions. Lack of PBANKA_122900 transcripts in the PBANKA_122900 KO clones was confirmed by RT-PCR, using RNA that was isolated from schizonts. The synthesis of cDNA is described in the transcript expression analysis section, and PCR was performed for 35 cycles of amplification using the oligonucleotides “e” plus “f” and “g” plus “h”.

Two fragments of 744 bp and 638 bp length corresponding to the gene PBANKA_114540 (shaded in gray in S4 Fig) were amplified by PCR using as template genomic DNA from P. berghei ANKA parasites and the primer pairs: PB106ko5SHindIII plus PB106ko5ASPstI; and PB106ko3SKpnI plus PB106ko3ASEcoRI. These PCR fragments were sequentially cloned into the restriction sites HindIII/PstI and KpnI/EcoRI of the plasmid pDEF-hDHFR. This gene targeting disruption plasmid was digested with HindIII and EcoRI to release the fragment for transfection. Transfection of P. berghei schizonts was performed as described for the PBANKA_122900 gene KO. Using this construct and gene disruption procedure we were unable to knockout PBANKA_114540 in 3 independent experiments.

Prepatent period and asexual multiplication of erythrocytic stages in vivo

To determine the prepatent period, 10² to 10⁴ salivary gland sporozoites from P. berghei ANKA wt or PBANKA_122900 KO lines were resuspended in RPMI and injected intravenously in the tail vein of naive 6 weeks old female Swiss Webster mice. At 24 h intervals after sporozoite injection, blood samples were withdrawn from the tail of injected animals and analyzed on Giemsa reagent-stained thin smears for the presence of parasites. The prepatent period was assessed by determining the number of days between sporozoite injection, and the time when at least 3 to 5 parasites could be detected by analyzing a minimum of 10,000 erythrocytes on Giemsa reagent-stained blood smears on glass slides. Two independent experiments were performed, using 5 mice per experiment. After prepatent period data collection, mice were euthanized.

To analyze the asexual multiplication of erythrocytic stages, 6 weeks old female Swiss Webster mice were injected intravenously with 2 x 10⁵ mature schizonts of either PBANKA_122900 KO or P. berghei ANKA wt parasites. Survival of the mice was monitored and parasitemia was determined by Giemsa reagent-stained thin blood smears collected at 24 h intervals. A similar experiment was performed by injecting 10³ and 10⁴ asexual stage parasites intravenously into 6 weeks old female C57Bl/6 mice. All experiments were performed twice with 5 mice per group per experiment. At the onset of clinical malaria symptoms mice were euthanized.

Sporozoite invasion and development assays

Hepa 1–6 cells were used for quantification of sporozoite invasion as described [32], using PBANKA_122900 KO or P. berghei ANKA wt sporozoites based upon double staining with a monoclonal antibody (mAb) 3D11, directed against the repeat region of P. berghei CSP in an assay that distinguishes intracellular from extracellular sporozoites [41, 42]. To quantify exoerythrocytic forms development PBANKA_122900 KO or P. berghei ANKA wt sporozoites were assayed for development in Hep 1–6 cells as described [32]. Experiments were performed in triplicate.

The P. falciparum phist gene family

The phist gene family was identified in the course of describing the repertoire of P. falciparum genes that encode proteins harboring PEXEL/HT motifs predicting export to the erythrocyte cytoplasm [12]. We revisited description of the published phist gene catalog and gene structures (see Fig 5 of Sargeant et al. [12]), in order to update the observation that 19 of the genes encoding PHIST domain proteins lacked predicted signal peptides and PEXEL/HT motifs, perhaps due to insufficient sequence information for gene models at the time of that publication. The current version of PlasmoDB (September 2015) matches our in-house annotation for all identified phist genes within the genome of the P. falciparum 3D7 isolate accessible at PlasmoDB and GenBank; which was arrived at via exhaustive iterative BLAST analyses, as well as annotation of all open reading frames within sub-telomeric regions and internal clusters of PEXEL/HT encoding genes. Indeed, the gene models for all 19 of the above genes were corrected, to show the presence of a signal peptide plus PEXEL/HT motif in 14 instances, and confirmation of the remaining genes as likely pseudogenes (compendia and details of methods in S1 Fig; detailed in S3 Table). Two of the 65 genes are possible pseudogenes (namely, MAL13P1.11 and PF14_0764), and additional predicted phist pseudogenes are listed in the inset of S1 Fig. The 7 resa-like genes were all confirmed [12] and no gene structure revisions were required, and no additional resa-like genes were identified. For both phist and resa-like genes 2-exon gene structures predominate, in which the signal peptide is encoded on the first exon and the PEXEL/HT motif is adjacent within the second exon. However, 6 phist genes are encoded by 3-exon gene structures, and 3- and 4-exon gene structures are also common in the predicted pseudogenes. A few of the phist genes are almost identical and might be indicative of subtelomeric blocks of duplicated DNA [2, 43]; for example, PFF0075c and PFF1510w on the left and right arms, respectively, of chromosome 6. In this study we did not revisit the nomenclature dividing the phist gene family into subfamilies, termed a, b and c [12], and such designations in S1 Fig.

A comparison of all PHIST proteins within the P. falciparum isolate nucleotide sequence databases (HB3, Dd2, RO-33/Ghana, D6, D10, VS/1, K1/Thailand, Santa Lucia, FCC-2/Hainan and Senegal V34.04; Broad Institute; downloaded for local TBLASTN analyses from the NCBI Trace Archive; as well as SNP data from the MalariaGen Pf3k project, www.malariagen.net/apps/Pf3k) indicates that the orthologous members from each isolate are 99–100% conserved (data not shown; [44]), despite the extensive sequence divergence between paralogs within the PHIST domain family, which typically possess less than 30% identity. This indicates that PHIST proteins likely diversified in response to a selective force other than immune pressure, as opposed to what is observed for the var genes, where orthologs are found to be highly divergent between field or laboratory isolates. This observation is in agreement with their predicted functional localization within the erythrocyte cytoplasm.

Transcript level analysis of P. falciparum phist and resa-like genes

To determine the transcript expression profiles of the phist and resa-like gene repertoire during the P. falciparum life cycle, we exploited the high-throughput gene expression data obtained for the asexual intraerythrocytic developmental cycle of P. falciparum accessible at PlasmoDB. Thirteen phist genes were selected as candidates for further studies, with the criteria of high expression levels but differing in the timing of expression, plus the 7 resa-like genes (available expression data shown in S2 Fig). Real time RT-PCR assays of these select genes yielded transcript expression profiles throughout the asexual intraerythrocytic developmental cycle (Fig 1) similar to the high-throughput transcriptome data, thus validating the expression patterns of the phist and resa-like genes described in PlasmoDB. For the 13 phist genes expression levels span a wide range and different stages of the asexual cycle, including transcription of a subset detectable in gametocytes [14]. This expression pattern might support the hypothesis of functional diversification of PHIST proteins, underpinned by multiple clusters of gene expression, where induction of subsets of PHIST genes occur at specific developmental stages (e.g. rings, trophozoites, and schizonts) when their products are required (examples are indicated by the red and blue highlighted genes in S2 Fig). We also compared the transcript levels of the select phist genes and the 7 resa-like genes in 4 isolates of P. falciparum, NF54, Dd2, HB3 and IT4 (Fig 2). Since the life cycle length differs slightly among these isolates, we chose to analyze late schizont stage parasites matched for similar developmental morphology in each isolate. All genes analyzed were expressed in the 4 isolates and their transcription levels at the schizont stage were sufficiently similar to indicate that expression of the PHIST and RESA-like protein repertoire is consistent in intraerythrocytic development across P. falciparum isolates.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 1. Transcript expression analysis of phist and resa-like genes during P. falciparum intraerythrocytic stages.

Gene-specific transcript levels for select phist genes A) and for the resa-like genes B) were analyzed by real time PCR using cDNA prepared from intraerythrocytic synchronized asexual and gametocyte P. falciparum cultures. Note that resa (PFA0110w) and resa2 (PF11_0509) are highly expressed relative to the other resa-like genes. Transcript expression was normalized to the expression of the control gene arginyl tRNA synthetase (PFL0900c). Hours post-synchronization indicate time in hours after adding purified schizonts to fresh blood culture. In the schematic depiction of phist and resa-like 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 C). The letter “G” indicates mature gametocytes.

https://doi.org/10.1371/journal.pone.0152510.g001

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 2. Transcript expression analysis of phist and resa-like genes in 4 P. falciparum isolates.

The transcript levels of select phist and resa-like genes in 4 isolates of P. falciparum, 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 arginyl tRNA synthetase (PFL0900c). In the schematic depiction of phist and resa-like genes, yellow represents the signal sequence, and red represents the PEXEL/HT motif.

https://doi.org/10.1371/journal.pone.0152510.g002

Protein structure, transcript level and cellular localization of two P. falciparum PHIST proteins

Two P. falciparum phist genes, PFE1600w and PFE1605w (the latter recently termed LyMP, [25]), indicated by orange highlight in S1 Fig, were selected for detailed characterization. The genes are located adjacent to each other within the subtelomeric region of the right arm of chromosome 5, and similarly to most phist genes, possess a typical 2-exon gene structure (Fig 3A). The PHIST domains of PFE1600w and PFE1605w each span approximately 150 aa and comprises 4 predicted interspersed alpha helices containing conserved tryptophan residues, which are hallmarks of all PHIST domains (Fig 3B). The unifying feature of PHIST domains is likely conserved structure, perhaps including key conserved residues, rather than primary amino acid sequence, as shown in Fig 3B. The 2 proteins share only 21% aa identity (Fig 3B), and it is this dissimilarity that motivated us to choose them for study.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 3. Protein structure of the phist genes PFE1600w and PFE1605w.

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.

https://doi.org/10.1371/journal.pone.0152510.g003

PFE1600w is predominantly transcribed in late ring and early trophozoite stages, while transcript levels of PFE1605w peak similarly but slightly later (Fig 1A). Low transcript levels of both genes can be detected soon after merozoite invasion and in late trophozoite and schizont stages, while transcripts were not observed in gametocytes.

As a means to understand PHIST protein function, we sought to determine the cellular localization and predicted export of PFE1600w and PFE1605w to the erythrocyte cytoplasm. Epitope-tagged constructs were designed to fuse 3 c-myc epitopes at the C-terminus of PFE1600w and PFE1605w (S3A Fig), and the resulting expression vectors were introduced into the P. falciparum NF54 isolate and selected for stable episomal expression via pyrimethamine drug pressure, as elaborated in the Materials and Methods section. Epitope-tagged PFE1600w and PFE1605w proteins were localized in synchronized asexual stages by indirect immunofluorescence assay (IFA) using anti-c-myc monoclonal antibodies. Epitope-tagged PHIST proteins were first observed in the ER of mid-ring stage of both PFE1600w and PFE1605w transformed parasite lines, in co-localization with the abundant ER resident protein, Pf39 (data not shown). At the late ring stage, PHIST proteins were observed exported to the erythrocyte cytoplasm, at least in partial co-localization with the marker of Maurer’s clefts, SBP1 (Fig 4A and 4B). PFE1600w was abundantly observed in the ER of late stage parasites (data not shown); and it is possible that this localization represents aberrant trafficking due to over-expression of the transcript and thereby protein, perhaps due to the use of a heterologous promoter. The localization to the erythrocyte cytoplasm validates trafficking that is predicted by the presence of a PEXEL/HT motif. Using a different fixation method, we also observed a broader distribution of a punctate pattern for PFE1605w-myc, having only partial overlap with the SBP1 marker (Fig 4C). PFE1605w was recently shown to be cotranslocated with PfEMP1 within the erythrocyte cytosol [13], in agreement with our localization data for PHIST proteins. Further study, of multiple PHIST proteins, is required to determine the association of PHIST protein with Maurer’s clefts. IFA on non-permeabilized infected erythrocytes failed to detect either of the epitope-tagged PHIST proteins on the erythrocyte surface (data not shown), suggesting a final cellular destination and function within the erythrocyte cytoplasm.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 4. Immunolocalization of c-myc epitope-tagged PFE1605w PHIST protein within infected erythrocytes.

A) 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) Co-localization was assayed using rabbit polyclonal SBP1 followed by goat anti-rabbit IgG conjugated with Alexa 594 (red). C) 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.

https://doi.org/10.1371/journal.pone.0152510.g004

Structure and transcript levels of P. berghei phist genes

P. falciparum and other Plasmodium species, such as P. reichenowi [45] P. vivax [12] and P. knowlesi have highly amplified sets of phist genes, but rodent malaria parasites have a minimal repertoire. In the P. berghei genome sequence we identified 3 phist genes (namely, PBANKA_114540, PBANKA_122900 and PBANKA_070080), via BLAST analysis using PHIST domains and pssm profiles as queries. Our result differs from the single P. berghei phist gene described in the original annotation [12]. Orthologs of PBANKA_114540 and PBANKA_122900 were identified in the genome nucleotide sequence databases for other rodent malaria parasites; namely, P. yoelii (PY00289 and PY01786, respectively) and P. chabaudi (PCAS_114490 and PCAS_122970, respectively). Additional phist genes were not identified in rodent malaria parasites. BLAST analyses using the PHIST domain as queries, identify as reciprocal best hits PBANKA_122900, the P. vivax PHIST protein PvPHIST/CVC-81₉₅ (PVX_093680), and P. falciparum PF08_0137. This indicates possible orthologous (vertically inherited) relationships, as recently proposed by Akinyi et al. [24]; and is supported by the observed synteny of adjacent genes, including the ookinete-expressed gene warp, together composing a locus which is conserved across the Plasmodium genus. PBANKA_122900 is internally localized in P. berghei, but the locus appears to be sub-telomeric in species other than rodent malaria parasites. Orthologous conservation across the Plasmodium genus might imply functional conservation underpinned by the PHIST domain, as discussed further in the Discussion section.

The P. berghei PHIST proteins PBANKA_114540 and PBANKA_122900 possess similar features to P. falciparum PHIST proteins; namely, signal peptides and PEXEL/HT trafficking motifs; and single PHIST domains, which are divergent in aa sequence with respect to each other (Fig 5A and 5B). Within the single predicted ORF of PBANKA_070080 we were unable to identify a signal peptide sequence, and there were no attractive upstream ORFs suggestive of an erroneous gene model. We thus propose that PBANKA_070080 is a pseudogene. The remaining P. berghei phist genes possess the typical 2-exon gene structure, in which the signal peptide is encoded on the first exon and the PEXEL/HT motif is located within the second exon (Fig 5A). To determine the transcript levels of the P. berghei phist genes throughout the parasite life cycle, quantitative real-time RT-PCR was performed using cDNA that was prepared from synchronized asexual stages, gametocytes and mosquito stage parasites (Fig 5C and 5D). PBANKA_114540 and PBANKA_122900 are predominantly transcribed in mature schizont stage parasites, and the transcript levels in schizonts were comparable to the levels of ama1 transcripts (PBANKA_091500), which was used as a positive marker for schizont stage transcription in addition to the control housekeeping gene, hsp70 (PBANKA_091441). Low expression of PBANKA_114540 was observed in gametocytes and no transcripts were detected in mosquito stages. Lack of detectable PBANKA_070080 transcripts supports the above annotation as a pseudogene, and accordingly our further analyses of phist genes did not include PBANKA_070080.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 5. The P. berghei phist 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.

https://doi.org/10.1371/journal.pone.0152510.g005

Cellular localization of P. berghei PHIST proteins

Immunolocalization studies using affinity purified rabbit polyclonal anti-sera against PBANKA_114540 and PBANKA_122900 revealed that both proteins are exported to the erythrocyte cytoplasm and observed throughout the intraerythrocytic life cycle (Fig 6A). In early asexual stages, both proteins exhibited a punctate, vesicle-like localization in the erythrocyte cytoplasm while in more mature stages the protein distribution appeared more diffuse. Immunofluorescence analysis also suggests that PBANKA_114540 and PBANKA_122900 associate with vesicle-like structures, or aggregations, in the erythrocyte cytoplasm (Fig 6A). In addition, the proteins co-localize, suggesting they share a common export system, or aggregation, in the rodent malaria parasites (Fig 6A). Staining of non-permeabilized cells was negative, indicating that PBANKA_114540 and PBANKA_122900 are not exposed on the surface of the infected erythrocyte (data not shown). We did not observe association of either PHIST protein with the erythrocyte membrane.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 6. Immunolocalization of P. berghei PHIST proteins PBANKA_114540, PBANKA_122900, and IBIS within infected erythrocytes.

A) 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) 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.

https://doi.org/10.1371/journal.pone.0152510.g006

To compare the punctate localization pattern of PHIST domain proteins with other P. berghei exported proteins that exhibit similarly punctate patterns, and to address possible co-localization within a single vesicle transport system, we analyzed the co-localization of PBANKA_136550 (IBIS1; [46]) and the PHIST protein PBANKA_122900 by IFA. We utilized a transgenic parasite line that expresses a mCherry-tagged version of PBANKA_136550. Analysis of live infected erythrocytes showed export of mCherry-PBANKA_136550 into the cytoplasm of the erythrocyte and localization in punctate vesicle-like structures (Fig 6B), as described for this protein by Ingmundson et al. [46]. Staining of mCherry-PBANKA_136550 parasites with anti-TER antibodies, a marker of the erythrocyte surface membrane, showed that PBANKA_136550 localizes exclusively within the erythrocyte cytoplasm (data not shown). Staining of fixed erythrocytes infected with mCherry-PBANKA_136550 parasites with anti-PBANKA_122900 antibodies showed a significant overlap between the fluorescence signals, indicating that both proteins localize within the same erythrocyte compartment (Fig 6B).

Functional analysis of P. berghei PHIST by targeted gene deletion

To address the function of PBANKA_114540 and PBANKA_122900 in P. berghei, we attempted to disrupt the genes via double-cross-over homologous recombination (S4 and S5A Figs, respectively). The PBANKA_114540 disruption construct was designed using the pDEF-hDHFR vector, while the PBANKA_122900 disruption construct used the vector b3D.DT^H.^D. The disruption vectors were transfected into P. berghei ANKA schizonts and transformants were selected via pyrimethamine drug pressure. In 3 independent transfection experiments we were unable to select mutant parasites with a disrupted PBANKA_114540 locus, suggesting that this gene is essential in asexual stage parasites. In contrast, deletion of PBANKA_122900 was achieved in a parallel experiment; as well as disruption of the PbTLP locus as a control, and correct disruption of these genes was verified by diagnostic PCR and Southern blot analysis (S5B and S5C Fig). Two PBANKA_122900 disruptant clones were selected for further characterization. Transcripts of PBANKA_122900 were not detected by RT-PCR in asexual stages of disruptant parasites (S5D Fig), confirming a successful deletion of the locus. The ability to generate disruptant parasites demonstrated that PBANKA_122900, as opposed to PBANKA_114540, is not essential for asexual intraerythrocytic development.

The PBANKA_122900 KO parasite lines showed normal gametocyte and ookinete formation in vitro (data not shown). Following transmission to mosquitoes the KO parasite lines produced oocysts that were morphologically indistinguishable from the wt control (S4 Table), and formed normal numbers of salivary gland sporozoites (S5 Table). Sporozoites from KO lines showed wt levels of hepatocyte invasion and developed into mature exoerythrocytic forms similarly to wt (S6 Fig). In addition, the infectivity of KO sporozoites in vivo was similar to wt, as shown by prepatent period after intravenous injection of 10² to 10³ sporozoites (S6 Table). Taken together, the assays employed in this study could not discern a role for PBANKA_122900 during development in the mosquito or the liver.

To describe the development of the asexual blood stages of PBANKA_122900 KO lines we determined the course of infection in Swiss Webster and C57Bl/6 mice following either inoculation with sporozoites or intraerythrocytic stages. An apparent trend towards long survival was observed in Swiss Webster mice (Fig 7A) or C57Bl/6 mice (Fig 7B) that were intravenously (i.v.) injected with 10⁴ and 10⁵ schizonts (Swiss Webster mice) or 10³ or 10⁴ mixed asexual stages (C57Bl/6 mice) of PBANKA_122900 KO parasites. However, the trend does not have statistical support. When we monitored the survival of mice after i.v. inoculation of 1,000 P. berghei ANKA wt or PBANKA_122900 k KO sporozoites, we observed a longer survival time of mice infected with PBANKA_122900 KO than those infected with wt parasites (Fig 7C), which is in support of the above mild growth delay of asexual blood stages. We did not detect differences in reticulocyte versus normocyte preference or a lower production of merozoites per schizonts; specifically, analysis of the percentage of young and mature erythrocytes infected with wt or KO parasites, at between 1 and 3% parasitemia before mice started producing reticulocytes, did not reveal a preference for invasion of one cell type by KO parasites (data not shown). In addition, erythrocytes infected with wt or KO parasites exhibited the same average number of merozoites per schizont (data not shown). In conclusion, further analysis of growth characteristics is required to discern any effects of disruption of PBANKA_122900 on the growth of asexual blood stages and lethality of KO parasites.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 7. Dynamics of malaria infection after inoculation with erythrocytic stage parasites and sporozoites.

Survival curves and daily parasitemia of A) Swiss Webster or B) C57Bl/6 mice injected intravenously with the indicated number of schizonts or mixed asexual stages of P. berghei 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 P. berghei 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.

https://doi.org/10.1371/journal.pone.0152510.g007