MOESM7 of In vivo compartmental kinetics of Plasmodium falciparum histidine-rich protein II in the blood of humans and in BALB/c mice infected with a transgenic Plasmodium berghei parasite expressing histidine-rich protein II

Additional file 7: Figure S4. Kinetics of recombinant PfHRP2 in plasma following protein injection. Female BALB/c mice (n = 3) were injected with rPfHRP2 protein. Plasma was collected at regular intervals post injection for quantification of rPfHRP2 plasma concentration. The protein half-life in each mouse was calculated and averaged using a first-order decay equation model constrained at a plateau of Y = 0. Data are represented as mean Âą SEM

HRPII-mediated vascular leakag<i>e</i> is blocked by antibody to IL-1β.

<p>Mice were infused as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0177142#pone.0177142.g001" target="_blank">Fig 1</a>, with HRPII plus an isotype antibody (Iso, positive control), HRPII plus anti-IL-1β antibody (experimental condition) or anti-IL-1β antibody alone (negative control). Untreated mice (no HRPII, no antibody) served as a further control. Vascular leakage in mice infused with HRPII/isotype is statistically significantly different from mice infused with HRPII/ anti-IL-1β, p = 0.01 (cerebellum) and p = 0.01 (cortex), by two-tailed t-test; p = 0.003 (cerebellum) and p = 0.06 (cortex) by ANOVA one-way variance with significance between HRPII/isotype and HRPII/ anti-IL-1β. Data are mean values +/-SEM for 6–12 mice per group accumulated over 3 independent experiments.</p

HRPII causes vascular leakage <i>in vivo</i>.

<p><b>(A)</b> Scheme of experimental design. Two doses of HRPII or BSA (200 μg) were injected into 4-week old female C57Bl/6 mice at 0 and 24 hours. At 48 hours, fluorescein levels in the cortex (<b>B</b>) and cerebellum (<b>C</b>) of the mice was measured. HRPII treatment was significantly different from control by two-tailed t-test, p = 0.01 (cortex) and p = 0.02 (cerebellum). Data are mean values +/-SEM for 8–16 mice per group accumulated over 3 independent experiments.</p

HRPII causes vascular leakage <i>in vivo</i>.

<p><b>(A)</b> Scheme of experimental design. Two doses of HRPII or BSA (200 μg) were injected into 4-week old female C57Bl/6 mice at 0 and 24 hours. At 48 hours, fluorescein levels in the cortex (<b>B</b>) and cerebellum (<b>C</b>) of the mice was measured. HRPII treatment was significantly different from control by two-tailed t-test, p = 0.01 (cortex) and p = 0.02 (cerebellum). Data are mean values +/-SEM for 8–16 mice per group accumulated over 3 independent experiments.</p

HRPII reduces survival time in an experimental cerebral malaria model.

<p>(<b>A</b>) Survival curves of 4-week old female mice infused with 50 μg of BSA or HRPII prior to infection with <i>P</i>. <i>berghei</i> ANKA (10⁵ parasites). Shown are the means for n = 24 to 27 mice pooled from four independent experiments. Curves are significantly different, p = 0.03, by the log-rank (Mantel-Cox) test. Mean time to death for HRPII = 11.5 days and for BSA = 16 days, p = 0.018, by two tailed t-test. (<b>B</b>) Mice displaying cerebral malaria-like symptoms died at low parasitemia by day 10, yet parasitemias between HRPII-infused mice and controls were closely matched on each day. Representative data from one of three experiments shown in panel A, 10 mice per group.</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

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

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

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

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