Table_1_The Consequences of Mixed-Species Malaria Parasite Co-Infections in Mice and Mosquitoes for Disease Severity, Parasite Fitness, and Transmission Success.XLSX

The distributions of human malaria parasite species overlap in most malarious regions of the world, and co-infections involving two or more malaria parasite species are common. Little is known about the consequences of interactions between species during co-infection for disease severity and parasite transmission success. Anti-malarial interventions can have disproportionate effects on malaria parasite species and may locally differentially reduce the number of species in circulation. Thus, it is important to have a clearer understanding of how the interactions between species affect disease and transmission dynamics. Controlled competition experiments using human malaria parasites are impossible, and thus we assessed the consequences of mixed-species infections on parasite fitness, disease severity, and transmission success using the rodent malaria parasite species Plasmodium chabaudi, Plasmodium yoelii, and Plasmodium vinckei. We compared the fitness of individual species within single species and co-infections in mice. We also assessed the disease severity of single vs. mixed infections in mice by measuring mortality rates, anemia, and weight loss. Finally, we compared the transmission success of parasites in single or mixed species infections by quantifying oocyst development in Anopheles stephensi mosquitoes. We found that co-infections of P. yoelii with either P. vinckei or P. chabaudi led to a dramatic increase in infection virulence, with 100% mortality observed in mixed species infections, compared to no mortality for P. yoelii and P. vinckei single infections, and 40% mortality for P. chabaudi single infections. The increased mortality in the mixed infections was associated with an inability to clear parasitaemia, with the non-P. yoelii parasite species persisting at higher parasite densities than in single infections. P. yoelii growth was suppressed in all mixed infections compared to single infections. Transmissibility of P. vinckei and P. chabaudi to mosquitoes was also reduced in the presence of P. yoelii in co-infections compared to single infections. The increased virulence of co-infections containing P. yoelii (reticulocyte restricted) and P. chabaudi or P. vinckei (predominantly normocyte restricted) may be due to parasite cell tropism and/or immune modulation of the host. We explain the reduction in transmission success of species in co-infections in terms of inter-species gamete incompatibility.</p

Conservation of <i>sbp1</i> gene synteny.

(A) Genomic information corresponding to 30 to 50 kb of the left arm of P. falciparum chromosome 5 (top), the right arm of P. knowlesi chromosome 10 (middle) and the left arm of P. berghei chromosome 11 (bottom). Syntenic genes are highlighted in orange. To better represent synteny, the P. knowlesi genome neighborhood is inverted, but the original color coding of genes encoded on the top strand (blue) and bottom strand (red) and genome position (represented by number scales for each species) have been retained. (B) Schematics of Plasmodium SBP1 orthologs showing the overall length and general protein structures. Repeat regions are shown in orange and the single transmembrane regions in light blue. Recently reported SBP1 orthologs of P. ovale and P. malariae are also included. (C) The conservation of amino acid sequence within the transmembrane and adjacent regions. The transmembrane regions are predicted by TMHMM 2.0 and highlighted in light blue.</p

Expression and localization of rPkSBP1 in infected monkey erythrocytes.

<p><b>(A)</b> Schematic of <i>P</i>. <i>knowlesi</i> rPkSBP1 expression construct (not to scale). Two myc epitopes (2myc) were fused at the C-terminus of full-length PkSBP1 open reading frame (PkSBP1 ORF) and expressed using the <i>P</i>. <i>falciparum</i> CRT 5' region (PfCRT 5') as a promoter. <b>(B)</b> Representative IFAT images of PkSBP1-transgenic <i>P</i>. <i>knowlesi</i> H-DMU line with anti-myc antibody (α-myc, green). α-myc-stained rPkSBP1 images were merged with DAPI nucleus-staining (blue) and differential interference contrast image (merge). The top panel is a negative control reacted with normal mouse IgG. R, ring; T, trophozoite; S, schizont stages. Scale bar represents 5 μm. <b>(C)</b> Western blotting of wild type parental <i>P</i>. <i>knowlesi</i> H-DMU line (WT) and PkSBP1-transgenic line (TG) with anti-myc antibody. Parasite proteins were sequentially extracted by freeze-thawing (FT), followed by extraction with 1% Triton X-100 (Tx), then with 2% SDS. Parasite protein cross-reacting by an antibody against <i>P</i>. <i>berghei</i> HSP70 serves as a loading control (bottom).</p

Expression and localization of rPk2TM-a in the monkey erythrocytes infected with Pk2TM-a-transgenic <i>P</i>. <i>knowlesi</i> H-DMU line.

<p><b>(A)</b> Schematic of the expression cassette of the <i>P</i>. <i>knowlesi</i> rPk2TM-a (not to scale). Two myc epitopes (2myc) were fused at the C-terminus of full-length Pk2TM-a open reading frame (Pk2TM-a ORF) and expressed using the <i>P</i>. <i>falciparum</i> CRT 5' region (PfCRT 5') as a promoter. Plasmid backbone is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164272#pone.0164272.g002" target="_blank">Fig 2A</a>. (<b>B</b>) Western blotting of wild type parental <i>P</i>. <i>knowlesi</i> H-DMU line (WT) and Pk2TM-a-transgenic <i>P</i>. <i>knowlesi</i> H-DMU line (TG) with anti-myc antibody (α-myc). Parasite proteins were sequentially extracted by freeze-thawing (FT), followed by extraction with 1% Triton X-100 (Tx), then with 2% SDS. Parasite protein cross-reacting by an antibody against <i>P</i>. <i>berghei</i> HSP70 serves as a loading control (bottom). <b>(C)</b> Representative IFAT images of Pk2TM-a-transgenic <i>P</i>. <i>knowlesi</i> parasites with anti-myc antibody (α-myc, green). α-myc-stained rPk2TM-a images were merged with DAPI nucleus-staining (blue) and differential interference contrast image (merge). The top panel is a negative control. R, ring; T, trophozoite; S, schizont stages. Scale bar represents 5 μm. <b>(D)</b> Representative transmission electron micrographs of immunogold labeled Pk2TM. Slit-like clefts (left) and oblong vesicular clefts (right) showed gold particles in the erythrocyte cytoplasm infected with Pk2TM-a-transgenic line. c, clefts; EM, erythrocyte membrane; P, parasite. Scale bar represents 500 nm.</p

rPkSBP1 is exported to <i>P</i>. <i>falciparum</i> Maurer's clefts and <i>P</i>. <i>knowlesi</i> ‘Sinton and Mulligan’ stipplings.

<p><b>(A)</b> Human erythrocytes infected with PkSBP1-transgenic <i>P</i>. <i>falciparum</i> co-stained with anti-myc antibody (green) and PfSBP1 (red). Merged image of rPkSBP1, PfSBP1, DAPI nucleus-staining (blue), and differential interference contrast (DIC) image are shown (merge). Top panel was labeled with anti-myc antibody (α-myc) and normal rabbit IgG, middle panel was labeled with normal mouse IgG and rabbit anti-PfSBP1 antibody, and bottom panel was labeled with mouse anti-myc and rabbit anti-PfSBP1 antibodies. <b>(B)</b> Colocalization of rPkSBP1 puncta (green) and Giemsa-stained ‘Sinton and Mulligan’ stipplings in monkey erythrocytes infected with PkSBP1-transgenic <i>P</i>. <i>knowlesi</i> H-DMU line. Merged image of rPkSBP1 and Giemsa-stained image are shown (merge). Scale bar represents 5 μm. Nuclei were stained with DAPI (blue).</p

PkSBP1 delineates <i>P</i>. <i>knowlesi</i> host modifications.

<p>Schematic representation of host erythrocyte modifications revealed by studying the localization of SBP1 ortholog. <i>P</i>. <i>knowlesi</i> infection in both monkey and human erythrocytes induces membranous structures onto which PkSBP1 localizes. Involvement of tether structures (white bars) adjoining these membranes to the host cell cytoskeleton is possible [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164272#pone.0164272.ref021" target="_blank">21</a>] but was not investigated in this study. PfSBP1 interacts with erythrocyte membrane protein 4.1 and spectrin, as described for <i>P</i>. <i>falciparum</i> (yellow and orange shapes) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164272#pone.0164272.ref016" target="_blank">16</a>]. PPM, parasite plasma membrane; PV, parasitophorous vacuole; PVM, parasitophorous vacuole membrane.</p

Expression and localization of rPkSBP1 in the human erythrocytes infected with PkSBP1-transgenic <i>P</i>. <i>knowlesi</i> H_hu-HSPH line.

<p><b>(A)</b> Representative fluorescence image showing localization of rPkSBP1 stained with anti-myc antibody (rPkSBP1, green) as puncta within the infected erythrocyte cytoplasm. rPkSBP1 signal was merged with erythrocyte membrane stained with anti-human CD235a (α-GlyA, red) and DAPI nucleus-staining (blue) (merge). DIC, differential interference contrast. Scale bar represents 5 μm. <b>(B)</b> Colocalization of rPkSBP1 puncta (green) and Giemsa stained ‘Sinton and Mulligan’ stipplings in the human erythrocyte infected with PkSBP1-transgenic <i>P</i>. <i>knowlesi</i> H_hu-HSPH line. Merged image of rPkSBP1 and Giemsa-stained image are shown (merge). Scale bar represents 5 μm. <b>(C)</b> Transmission electron micrographs of two representative erythrocytes infected with PkSBP1-transgenic <i>P</i>. <i>knowlesi</i> H_hu-HSPH line. Slit-like clefts (left) and oblong vesicular clefts (right) were observed. c, clefts; cv, caveola; EM, erythrocyte membrane; P, parasite. Scale bar represents 500 nm.</p

rPkSBP1-positive membranous structures in the monkey erythrocytes infected with PkSBP1-transgenic H-DMU line.

<p>Representative micrographs of immunogold-labeled rPkSBP1. Gold particles were visible at slit-like clefts (<b>A</b>) and oblong vesicular clefts (<b>B</b>) in the erythrocyte cytoplasm infected with PkSBP1-transgenic line. c, clefts; EM, erythrocyte membrane; P, parasite. Scale bar represents 500 nm.</p

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

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