TRAP-DMut sporozoites are severely impaired in gliding motility and host cell invasion.

<p>(A) Gliding motility of TRAP-JMD and TRAP-DMut sporozoites. Salivary gland sporozoites were incubated on slides for 1 hr and trails were visualized and counted. The percentage of sporozoites with and without trails is shown in the pie charts. For those sporozoites associated with trails, the number of circles produced by each sporozoite was counted and shown is their distribution for each parasite line. Over 100 sporozoites per well were counted and shown are the means of triplicate wells ± SD. (B) Representative images of the types of trails produced by each mutant. (C) Hepatocyte invasion. Salivary gland sporozoites were incubated with Hepa 1–6 cells for 1 hr, fixed and stained with a double staining assay that distinguishes extracellular and intracellular sporozoites. Percent invasion was determined and shown are the means ± SD of duplicate wells. All experiments were performed at least twice and shown is a representative experiment.</p

ROM4 is expressed on the sporozoite surface and antibodies against the extracellular tail of ROM4 inhibit hepatocyte invasion.

<p>(A) Western blot analysis of <i>P. berghei</i> erythrocytic stage schizont lysate (sch) and salivary gland sporozoites (spz) probed with anti-PfROM4 C-terminal IgG. Molecular weight markers are in kDa. (B) Immunofluorescence of <i>P. berghei</i> salivary gland sporozoites fixed with cold methanol and stained with anti-CSP antibodies and anti-PfROM4 C-terminal IgG.</p

Impaired TRAP processing of rhomboid cleavage site mutants leads to impaired host cell invasion.

<p>(A) In vitro invasion. Salivary gland sporozoites were incubated with Hepa 1–6 cells and fixed after 1 hr (data on left) or 6 hrs (data on right). Cells fixed after 1 hr were stained with a double staining assay that distinguishes between extracellular and intracellular sporozoites and the percent of total sporozoites that were intracellular was determined (left axis). Cells fixed after 6 hrs were stained with UIS4 antisera to determine the number of sporozoites that had entered in a vacuole (right axis). For both experiments at least 50 fields per well were counted and shown are the means ± SD of duplicate wells. (B) Kinetics of entry into hepatocytes. Salivary gland sporozoites were incubated with Hepa 1–6 cells for 15, 30 and 45 mins before being washed, fixed and stained with a double-staining assay that distinguishes extracellular and intracellular sporozoites. Shown is the percent of total sporozoites that were in the process of entering host cells, i.e. partially inside and partially outside. 50 fields per coverslip were counted and the means of duplicates ±SD are shown. (C) EEF development. Salivary gland sporozoites were added to Hepa 1,6 cells and incubated for 48 hrs at which time they were fixed and stained. The number of EEFs in 50 fields per coverslip were counted and shown are the means ± SD of duplicate wells. (D) Cell traversal. Salivary gland sporozoites were incubated with Hepa 1,6 cells for 1 hr, in the presence of the nucleic acid dye TOTO-1. Controls were pre-incubated and kept in the presence of cytochalasin D (CD), which inhibits motility. The number of TOTO-1 positive cells in 50 fields was counted and the means ±SD of duplicate wells are shown. All experiments were performed at least twice and representative experiments are shown.</p

In vivo infectivity of TRAP mutant sporozoites as determined by prepatent period.

<p>In vivo infectivity of TRAP mutant sporozoites as determined by prepatent period.</p

Disruption of the rhomboid motif impairs TRAP cleavage.

<p>(A) Primary structure of TRAP expressed in TRAP-VAL and TRAP-FFF mutants, with the point mutations introduced to disrupt the putative rhomboid substrate motif indicated above each transmembrane domain. (B) Western blot analysis of recombinant control TRAP-rWT (rWT) and rhomboid cleavage mutant salivary gland sporozoites TRAP-VAL and TRAP-FFF (VAL and FFF) probed with TRAP anti-repeat antisera. As a loading control the bottom half of the membrane was probed with mAb 3D11 which recognizes the repeat region of CSP. (C) Pulse-chase metabolic labeling of TRAP-rWT and rhomboid cleavage site mutants. Salivary gland sporozoites were metabolically labeled for 1 hr and either placed on ice for 4 hrs (Time = 0) or chased for 4 hrs (Time = 4). Sporozoites were then centrifuged and TRAP was immunoprecipitated from either the pellet (P) or supernatant (S) using anti-repeat antisera and analyzed by SDS-PAGE and autoradiography. Molecular weight markers shown on left of top panel. Top panel: 6 day exposure. Bottom panel: 14 day exposure, arrows show location of processed VAL and FFF mutant TRAP. (D) Immunofluorescence analysis of surface TRAP staining in TRAP-rWT and rhomboid cleavage site mutants. Shown are representative fluorescence and phase contrast images of the TRAP staining pattern after fixation with paraformaldehyde. Microscope and camera settings were identical for all photographs. (E) Box plot of fluorescence intensity of TRAP surface staining in TRAP-rWT and rhomboid cleavage site mutants. Unpermeabilized sporozoites were stained with anti-TRAP repeat antisera and intensity of staining was measured using NIS Elements software. Identical camera and microscope settings were used for all measurements. Boxes contain 50% of the data around its median (black line in box). Whiskers show the range of data within the 10^th and 90^th percentiles and outliers are shown individually. Results are pooled from 2 to 4 independent experiments. There was a statistically significant difference in staining intensity between TRAP-rWT and TRAP-VAL sporozoites (p<.0001) and between TRAP-rWT and TRAP-FFF sporozoites (p<.0001).</p

Impaired TRAP processing leads to aberrant gliding motility.

<p>(A) Gliding motility of rhomboid cleavage site mutants. Salivary gland sporozoites were incubated on slides for 1 hr and trails were visualized and counted. The percentage of sporozoites with and without trails is shown in the pie charts. For those sporozoites associated with trails, the number of circles produced by each sporozoite was counted and shown is their distribution for each parasite line. Asterisks indicate that none of the TRAP-VAL and TRAP-FFF mutants were associated with over 50 circles. Over 100 sporozoites per well were counted and shown are the means of triplicate wells ± SD. (B) Live imaging of gliding motility of rhomboid cleavage site mutant sporozoites. Sporozoites were observed and recorded using a Leica laser scanning confocal microscope. Time lapse images of sporozoites gliding on glass bottom dishes are shown with the maximum intensity projection on the right. (C) For each parasite line the average speed of ten sporozoites was determined for 60 s. All experiments were performed at least twice and a representative experiment is shown.</p

Sporozoite numbers and localization in mosquitoes infected with control and mutant parasites.

<p>*Mosquitoes were infected with the indicated parasite clones and midguts, hemolymph and salivary glands were harvested from 15 mosquitoes at days 14, 16 and 18 post-infective blood meal respectively, pooled and sporozoites were counted. Shown are the means of three independent experiments ± SD.</p>#<p>Sporozoites in the supernatant and pellet of trypsin-treated salivary glands were counted and the percentage inside was calculated. There were 15 mosquitoes per group. This experiment was performed twice with similar results.</p

TRAP is proteolytically processed and shed from the sporozoite surface by a serine protease.

<p>(A) Primary Structure of TRAP: Shown are the extracellular adhesive domains, namely the A-domain and the type I thrombospondin repeat (TSR), as well as the repeat region, the juxtamembrane region (JMD), the transmembrane domain (TM) and cytoplasmic tail (CT). Anti-TRAP antibodies used in this study recognize either the repeat region (α-Rep) or the cytoplasmic tail of TRAP (α-CT). (B) Pulse-chase metabolic labeling and TRAP immunoprecipitation using anti-repeat or anti-cytoplasmic tail antisera. Salivary gland sporozoites were metabolically labeled and placed on ice for 2 hrs (Time = 0) or chased at 28°C for 2 hrs (Time = 2). Sporozoites were then centrifuged and TRAP was immunoprecipitated from either the pellet (P) or supernatant (S) using antibodies against the repeat region of TRAP (left panel) or antibodies against the cytoplasmic tail (right panel) and analyzed by SDS-PAGE and autoradiography. Supernatants from control sporozoites kept on ice did not contain any TRAP (data not shown). (C) Effect of protease inhibitors on TRAP cleavage. Salivary gland sporozoites were metabolically labeled and chased at 28°C for 2 hrs in the presence of the indicated protease inhibitors. Sporozoites were then centrifuged and TRAP was immunoprecipitated from either the pellet (P) or supernatant (S) using anti-repeat antisera and analyzed by SDS-PAGE and autoradiography. The following inhibitors were used: 10 µM E64, 1 mM PMSF, 1 µM pepstatin (Pep), 0.3 µM aprotinin (Apr), 100 µM 3,4 DCI, 100 µM TLCK, 75 µM leupeptin (Leu), and 5 mM EDTA. (D) Effect of protease inhibitors on gliding motility. Salivary gland sporozoites were pre-incubated with the indicated protease inhibitors and then added to slides in the continued presence of the inhibitor for 1 hr at 37°C. Sporozoite trails were visualized and the number of sporozoites with and without trails was counted. Inhibition of motility was calculated based on the motility of sporozoites pre-treated with media alone. Each inhibitor was tested in triplicate and 50 fields per well were counted. The means ± SD are shown. DCI-R indicates that DCI was replenished every 20 min. All inhibitors were tested in at least two independent experiments however DCI and PMSF were tested in 3 or more independent experiments. A representative experiment is shown.</p

BCKDH is required for growth of <i>Plasmodium berghei</i> in mature RBCs.

<p>(A) Total lysates from a mixed population of parasitic stages for <i>P. falciparum</i> 3D7 (Pf), Pb wild type (WT) and Pb<i>e1a_ko</i> were analysed by Western blot. Expression of BCKDH-E1a (shown by the arrow) was assessed using cross-reacting anti-PfBCKDH-E1a antibodies. Profilin was used as loading control. (B) Parasitaemia was followed daily in mice infected with WT (blue line) or Pb<i>e1a_ko</i> (red line). Each line corresponds to the parasitaemia of one mouse. 5 mice were infected per condition. (C) Haematocrit was followed over the course of infection in mice infected with WT (blue) or Pb<i>e1a_ko</i> (red). Corresponding parasitaemia levels of this experiment are shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004263#ppat.1004263.s003" target="_blank">Fig. S3B</a>. The dotted line represents the mean haematocrit level of uninfected control mice followed throughout the experiment. Data show mean ± SD from 4 mice per condition. (D) Parasitaemia was followed daily in mice pre-treated (full lines) or not (dotted lines) with phenylhydrazine to induce reticulocytosis 3 days prior infection with parasites. Mice were infected with 5×10⁶ WT (blue line) or Pb<i>e1a_ko</i> (red line) parasites. 5 mice were infected per condition and lines represent mean parasitaemia ± SD. (E) Invasion of Pb<i>e1a_ko</i> (red) compared to WT (blue). Vybrant Green-labeled purified schizonts were incubated with DDAO-SE-labeled purified normocytes or reticulocytes, and free merozoites were allowed to invade. Parasitaemia in the DDAO-SE-labeled target population was determined by flow cytometry. Invasion efficiency was determined as a percentage of the WT control parasites. Cytochalasin D (CD)-treated schizonts were used as a negative control. Data are represented as mean ± SD of three independent biological replicates. (F) Giemsa-stained blood smears showing normal development of WT, Pb<i>e1a_ko</i> and complemented Pb<i>e1a_ko</i>+Pfe1a parasites to the schizont stage in purified reticulocytes. Parasites were cultured <i>in vitro</i> for the times indicated. (G) Giemsa-stained blood smears showing development of the different strains within purified normocytes. WT parasites mature normally from ring to schizont stage while Pb<i>e1a_ko</i> degenerate rapidly. Complemented Pb<i>e1a_ko</i>+Pfe1a restored the ability of Pb<i>e1a_ko</i> to develop within normocytes. Parasites were cultured <i>in vitro</i> for the times indicated.</p

<i>Toxoplasma gondii</i> BCKDH-complex is required for normal growth and virulence.

<p>(A) Schematic representation of pathways to produce acetyl-CoA in the mitochondrion. In green are pathways specific to <i>T. gondii</i> and in red pathways common to <i>T. gondii</i> and <i>Plasmodium</i> spp. (B) Total lysates from extracellular RH<i>ku80_ko</i> (RH) and Tg<i>e1a_ko</i> tachyzoites were analysed by Western blot. Expression of BCKDH-E1a was assessed using polyclonal anti-PfBCKDH-E1a antibodies. Detection of profilin was used as loading control. (C) Plaque assays were performed by inoculating HFF monolayers with RH or Tg<i>e1a_ko</i> parasites for 7 days. Plaques were revealed by Giemsa staining of HFFs. Scale bar represents 1 mm. (D) Intracellular growth of RH (blue) and Tg<i>e1a_ko</i> (red) was assessed after 24 h in complete media, media lacking glutamine, or glucose. Following 24 h of growth in glucose-depleted environment, glucose was added back to the media and rescue of the parasite’s growth was assessed. Data are represented as means ± SD from three independent biological replicates. (E) The apicoplast targeting sequence of TgPDH-E1a (aa 1–225, ABE76506) and mitochondrial targeting sequence of TgBCKDH-E1a (aa 1–73, XP_002366588) were replaced with the mitochondrial transit peptide of the superoxide dismutase 3 (SOD3) and myc-tagged <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004263#ppat.1004263-Pino2" target="_blank">[71]</a> to direct the expression of the fusion protein in the mitochondrion of Tg<i>e1a_ko</i> parasites for complementation (creating pTub8-SOD3mycPDHE1a and pTub8-SOD3mycBCKDHE1a respectively). Immunofluorescence assay shows localization of SOD3mycPDHE1a and SOD3mycBCKDHE1a in the single tubular mitochondrion (anti-myc (in green), anti-GAP45 (pellicle marker in red)). (F) Intracellular growth assay at 32 h post transient transfection of Tg<i>e1a_ko</i> with pTub8-SOD3mycPDHE1a, pTub8-SOD3mycBCKDHE1a and pTub8-mycNtGAP45 (negative control) in complete media or media depleted in glucose. Data are represented as means ± SD from three independent biological replicates. Only vacuoles containing parasites transiently expressing the transgene were taken into account. Over 200 vacuoles were counted per replicate. (G) CD1 mice were infected with RH (in blue) or Tg<i>e1a_ko</i> (in red) tachyzoites (∼15 parasites per mouse) and survival was assessed over 21 days. A challenge with ∼1000 wild-type RH tachyzoites was performed on mice that survived initial infection and survival followed for a further 10 days. Five mice were infected per condition.</p