Proposed role of <i>P. falciparum</i> Hsp110 and other chaperones as capacitors for evolutionary change.

<p>Malaria parasite proteins with asparagine repeat–containing sequences have a greater risk of aggregation. <i>P. falciparum</i> Hsp110c, possibly with the help of other chaperones, negates much of this risk, thereby allowing these loop-like regions to mutate. Over time these mutations can give rise to novel protein domains, allowing the parasite to develop new functionalities such as drug resistance and new pathogenic factors.</p

<i>Plasmodium</i> parasites require heme as a metabolic cofactor throughout their life cycle.

<p><i>Plasmodium</i> enzyme abbreviations are consistent with those used at the <i>Plasmodium</i> Genomics Resource (PlasmoDB.org). Abbreviations: ALAD, aminolevulinic acid dehydratase; ALAS, aminolevulinic acid synthase; CPO, coproporphyrinogen oxidase; FC, ferrochelatase; PBGD, porphobilinogen deaminase; PPO, protoporphyrinogen oxidase; UROD, uroporphyrinogen decarboxylase; UROS, uroporphyrinogen synthase.</p

Ablating heme biosynthesis has no effect on blood-stage <i>P</i>. <i>falciparum</i> growth.

<p>(A) Mass spectra (from [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006511#ppat.1006511.ref012" target="_blank">12</a>]) for detection of [¹³C]heme biosynthesized from ¹³C₄-ALA in WT <i>P</i>. <i>falciparum</i> D10 parasites, ΔFC parasites, or WT parasites grown in 200 μM SA. Spectra were normalized to the intensity of the heme peak in the WT parasite sample and offset to avoid baseline overlap. (B) Growth of asynchronous WT or ΔFC D10 parasites in the absence or presence of 200 μM SA (from [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006511#ppat.1006511.ref013" target="_blank">13</a>]). Measured parasitemia values for each parasite line were normalized to the respective parasitemia on day 4 and fit with an exponential growth equation. Abbreviations: ΔPfFC, genetic knockout of P. falciparum ferrochelatase; SA, succinylacetone; WT, wild-type.</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-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 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-Cell Enzymology To Probe His–Heme Ligation in Heme Oxygenase Catalysis

Heme oxygenase (HO) is a ubiquitous enzyme with key roles in inflammation, cell signaling, heme disposal, and iron acquisition. HO catalyzes the oxidative conversion of heme to biliverdin (BV) using a conserved histidine to coordinate the iron atom of bound heme. This His–heme interaction has been regarded as being essential for enzyme activity, because His-to-Ala mutants fail to convert heme to biliverdin <i>in vitro</i>. We probed a panel of proximal His mutants of cyanobacterial, human, and plant HO enzymes using a live-cell activity assay based on heterologous co-expression in <i>Escherichia coli</i> of each HO mutant and a fluorescent biliverdin biosensor. In contrast to <i>in vitro</i> studies with purified proteins, we observed that multiple HO mutants retained significant activity within the intracellular environment of bacteria. X-ray crystallographic structures of human HO1 H25R with bound heme and additional functional studies suggest that HO mutant activity inside these cells does not involve heme ligation by a proximal amino acid. Our study reveals unexpected plasticity in the active site binding interactions with heme that can support HO activity within cells, suggests important contributions by the surrounding active site environment to HO catalysis, and can guide efforts to understand the evolution and divergence of HO function

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

DPAP3 has proteolytic activity.

<p>(<b>A</b>) Analysis of purified rDPAP3. Two main bands are detected by silver stain, both of which are strongly labelled by FY01 and recognized by the anti-Nt-DPAP3 and anti-Ct-DPAP3 antibodies. All other minor bands in the silver stain are also recognized by DPAP3 antibodies and represent degradation products that could not be separated during purification. (<b>B</b>) Measurement of VR-ACC turnover and FY01 labelling for WT and C504S MUT rDPAP3. Silver stain analysis shows equivalent amounts of protein were obtained from the purification of WT and MUT rDPAP3. (<b>C</b>) pH dependence of rDPAP3 activity measured at 10 μM VR-ACC (n = 3).</p

Role of DPAP3 in RBC invasion.

<p>(<b>A</b>) Representative FACS plot (forward light scattering vs. Hoechst staining) showing a decrease in invasion of the A1cKO upon RAP treatment. The populations of uRBCs, rings, and schizonts (Sch) are indicated. (<b>B</b>) Analysis of invasion efficiency of DPAP3 cKO and complementation lines. Schizonts collected 45 h after DMSO or RAP treatment were incubated with fresh erythrocytes for 8–14 h, fixed, stained with Hoechst, and analyzed by FACS. Shown is the ratio in invasion efficiency between RAP- and DMSO-treated parasites. Filled and empty circles represent individual biological replicates for the F8cKO and A1cKO, respectively and their corresponding complementation lines. Student’s t test significance values between cell lines are shown above the lines, or above each bar when comparing to the E7ctr. Only significant p-values are shown. (<b>C</b>) FACS analysis of extracellular merozoites. C2-arrested A1cKO schizonts pretreated with DMSO or RAP were incubated with fresh RBCs after C2 removal. Samples were collected at the indicated time points, fixed, and stained with Hoechst and WGA-Alexa647. The FACS plot and histogram show samples collected 20 min after C2 washout. Free merozoites (Mrz) show positive staining for DNA but negative for WGA-Alexa647. Quantification of the different parasite stage populations over time is shown on the bar graph; biological replicates are shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007031#ppat.1007031.s013" target="_blank">S9 Fig</a>. (<b>D</b>) Quantification of attached merozoites by flow cytometry. Samples collected 15 and 20 min after C2 washout during invasion assays (performed as in <b>C</b>), were stained with Hoechst and anti-MSP1 antibody (anti-mouse Alexa488 as secondary antibody). Because MSP1 is shed during invasion, merozoites attached to the RBCM (Att Mrz) can be differentiated from intracellular parasites as the cell population positive for DNA and MSP1 staining. FACS plots compare anti-mouse Alexa488 staining in samples treated with or without the anti-MSP1 antibody. MSP1 staining (green) of attached merozoites under these conditions was confirmed by microscopy (central panel). Quantification of the population of attached merozoites relative to the ring population is shown on the bar graph. Circles represent different biological replicates: filled for F8cKO and empty for A1cKO. No significant difference was observed between DMSO and RAP treatment.</p