Journal: Blood

Article Title: Remodeling of the malaria parasite and host human red cell by vesicle amplification that induces artemisinin resistance

doi: 10.1182/blood-2017-11-814665

Figure Lengend Snippet: Stage-specific expression and localization of K13 in relation to ER-PI3P, PfBiP, and PfEMP1. (A) Custom antibodies to K13 (supplemental Methods) detect an 83-kDa band in Pf3D7-infected red cells (IRs) but not uninfected red cells (URs) in Western blots (molecular weights in kDa) and localize K13 (green) by IFA in trophozoite and schizont stages (counterstained for host band 3, red), as imaged with a 100×, NA-1.4 objective on an Olympus IX inverted fluorescence microscope using DeltaVision Deconvolution microscopy.25 (B-C) Cryo-IEM of PfNF54K13WT dually probed for PI3P (6 nm gold) and ER marker BiP (15 nm gold) (B) or K13 (15 nm gold) (C). Black arrow indicates PI3P in lumen of ER tubule; yellow arrows indicate cytoplasmic PI3P; double yellow arrows indicate PI3P vesicular clusters outside of ER tubules on the cytoplasmic face; red arrow indicates low level of PI3P vesicles devoid of K13. Scale bar, 100 nm. (D) Membrane association of K13. Lysates of Pf3D7 were treated as indicated, separated by centrifugation (15 000 g for 30 min) into membrane pellet and soluble supernatant fractions and probed in Western blots for parasite and host (human) markers. Adding 6 M urea (a strong chaotropic agent) for 30 min at 23°C to parasite cell lysates failed to release K13 from the pellet (of parasite cell lysates), although the cytosolic parasite protein PfFKBP was quantitatively detected in the soluble fraction, suggesting that K13 was membrane associated. Sodium carbonate 100 mM, pH 11.5, for 30 min on ice released K13 from the pellet, suggesting that it was peripherally (but not integrally) associated with membranes (and consistently, K13 was also released by 1% Tx-100 for 30 min at room temperature or 1% SDS for 30 min at room temperature). Human band 3 was only released by 1% Triton or 1% SDS, confirming that it was integrally membrane associated. Molecular weight standards (in kDa) are as shown. (E) Stereological analyses of K13-gold particle distribution by cryo-IEM. Vesicles close to the ER appear to bud from ER tubules. Vesicles distal to the ER may be derived from other organellar membranes and cannot be ascribed solely to the ER. (F) IFA single optical sections localizing K13 in segmenter, merozoite, and ring stages. Scale bars are as shown. (G) Anti-ATS antibodies recognize a band >250 kDa in Western blots (as was expected for PfEMP1) in IRs but not URs. Molecular weight standards (in kDa) are as shown. (H) IFA showing single optical sections colocalizing K13 and PfEMP1 (labeled by anti-ATS) in Pf3D7 segmenter and merozoites. Pearson’s correlation coefficients are as indicated. Experimental replicates, n = 3. Scale bars are as indicated. Parasite nucleus (blue) is stained with Hoechst 33242. HuBand3, Human band 3; P, pellet; PC, Pearson’s correlation coefficient; S, supernatant.

Article Snippet: Pan-“PfEMP1” antibodies were raised to recombinant conserved C-terminal acidic-terminal sequence (ATS) domain of PfEMP1 by the commercial vendor Genscript Inc. Anti-PI3P was from Echelon Biosciences.

Techniques: Expressing, Infection, Western Blot, Fluorescence, Microscopy, Marker, Membrane, Centrifugation, Molecular Weight, Derivative Assay, Labeling, Staining

Journal: Blood

Article Title: Remodeling of the malaria parasite and host human red cell by vesicle amplification that induces artemisinin resistance

doi: 10.1182/blood-2017-11-814665

Figure Lengend Snippet: K13 copurified in a PfEMP1-immunoproteome enriched in proteostatic pathways that include the UPR, implicated in clinical artemisinin resistance. (A) Detection of PfEMP1 (arrow, left panel) and total protein content (right panel) captured by anti-ATS antibodies (but not in its absence at −1°) on covalently conjugated protein G beads. Molecular weight standards are in kDa. *Heavy IgG; **Light IgG. (B) Intensity properties of 2 replicate PfEMP1 immunoproteomes. (C) Distribution of proteins in the “503-proteome” comparing their abundance in each proteome expressed as a ratio (abundance ratio [AR] = abundance in Proteome 1/abundance in Proteome 2). X-axis: AR interval range; y-axis: number of proteins. (D) Hypergeometric analyses of 503-proteome and artemisinin-resistant transcriptome associated with clinical strains12 (for proteostasis enrichment and more detail, see supplemental Tables 2-4; Figure 3E-G). (E) Distribution of normalized intensity (ni; intensity divided by molecular weight) of the 207-proteome (AR = 0.5-1.99). Reactive oxidative stress complex components are BiP and PDI-11 (ni 5.17e8 and 1.2e7, respectively; supplemental Table 5). For detailed annotation and proteostasis enrichment, see supplemental Table 4 and supplemental Figure 3H-I. (F) Model integrating K13-PI3P tubules/vesicles (black-gray spheres based on findings of Figures 1 and ​and22 and supplemental Figures 1 and 2) and K13-PfEMP1 immunoproteome (green; based on findings in Figure 3; supplemental Figure 3) in proteostatic mechanisms of vesicular export and protein quality control and folding in the ER and cytoplasm (dotted circle). Experimental replicates, n = 2. Other organelles and key are as shown. HT, host targeting sequence; PEXEL, plasmodium export element; PNEP, PEXEL-negative proteins; PPM, parasite plasma membrane; PVM, parasitophorous vacuolar membrane; ROSC, reactive oxidative stress complex.

Article Snippet: Pan-“PfEMP1” antibodies were raised to recombinant conserved C-terminal acidic-terminal sequence (ATS) domain of PfEMP1 by the commercial vendor Genscript Inc. Anti-PI3P was from Echelon Biosciences.

Techniques: Molecular Weight, Control, Sequencing, Clinical Proteomics, Membrane

Journal: Blood

Article Title: Remodeling of the malaria parasite and host human red cell by vesicle amplification that induces artemisinin resistance

doi: 10.1182/blood-2017-11-814665

Figure Lengend Snippet: Synthetic elevation of PI3P that induces resistance in absence of K13 mutation yields vesicle immunoproteomes enriched in signatures of proteostasis and clinical resistance. (A-B) Detection of PfEMP1 (arrow) captured by anti-ATS antibodies (but not in its absence; −1°) from PfVPS34myc and mass intensity properties of 2 replicate immunoproteomes. (C-D) Detection of PfEMP1 (arrow) captured by anti-ATS antibodies (but not in its absence; −1°) from immunoproteome from PfVPS34AAAmyc (catalytic dead enzyme in which AAA replaces the VPS34-catalytic active-site residues glutamic acid, arginine, and histidine DRH) and mass intensity properties of 2 replicate proteomes. Molecular weight standards are in kDa. (E) Comparative distribution of proteins in proteomes of VPS34myc and VPS34AAAmyc. (F) Hypergeometric analyses showing enrichment of VPS34myc immunoproteome and upregulated (but not downregulated) transcripts of the clinical artemisinin-resistant transcriptome.12 (G) Protein distribution analyses indicating that 95% of Pf3D7 of proteins enriched in the clinical transcriptome are present in the PfVPS34myc PfEMP1 immunoproteome. (H) Number and percentage of hits for indicated chaperone networks in the vesicle immunoproteome from PfVPSmyc reveals substantial association with reactive oxidative stress complex and T-complex chaperones also known as TCP1 ring complex of the UPR. Mu, mass units.

Article Snippet: Pan-“PfEMP1” antibodies were raised to recombinant conserved C-terminal acidic-terminal sequence (ATS) domain of PfEMP1 by the commercial vendor Genscript Inc. Anti-PI3P was from Echelon Biosciences.

Techniques: Mutagenesis, Molecular Weight

Journal: Blood

Article Title: Remodeling of the malaria parasite and host human red cell by vesicle amplification that induces artemisinin resistance

doi: 10.1182/blood-2017-11-814665

Figure Lengend Snippet: Effect of K13 mutation and drug exposure on PfEMP1 expression and export. (A) Schematic of parasite drug exposure. (B) RNA sequence analyses of PfEMP1 expression in 0- to 3-hour PfNF54K13WT and PfNF54K13C580Y rings exposed to 4 nM DHA (positive, red) or mock treated (negative, blue) for 6 hours. Intensity units are as follows: y-axis, PfEMP1 gene id; x-axis, black arrows indicate a major transcript seen in WT parasites. The main PfEMP1 transcript 1200600 (var2csa) was expressed in both wild-type and mutant parasites +/−DHA. Second and third K13WT transcripts (0425800, 0712300) were also expressed in DHA. In C580Y, 0425800, 0400400, 0600200, 0800200, and 0300300 were expressed +/−DHA. PfEMP1 transcript levels were sustained an hour after DHA was washed out (supplemental Figure 5A-B), and parasites successfully matured through subsequent stages of the asexual life cycle (not shown). (C-D) DHA 4 nM potently inhibits PfEMP1 export (green; detected by ATS antibodies in IFA) to the red cell in artemisinin-sensitive PfNF54K13WT but not resistant PfNF54K13C580Y (C). Quantitative analyses of 400 optical sections through 30 infected red cells (D). (E-F) Quantitative analyses of sensitivity of PfEMP1 export (green) to DHA in the artemisinin-sensitive clinical strain ANL-1 but not its resistant clinical counterpart ANL-2 (C580Y). Western blots reveal equivalent PfEMP1 protein levels with or without DHA. For all samples, pixel intensity at 100% exceeded 6.5 × 106 (AU) (panels C-F). Experimental replicates: n = 2 (panels A-B); n = 2 (panels C-F). PfExp2 (Pf-exported protein 2, red), a parasitophorous vacuolar membrane maker, was used to stain the parasite periphery (panels C,E). Scale bars, 5 µm. Imaged with a 100×, NA-1.4 objective on an Olympus IX inverted fluorescence microscope using DeltaVision Deconvolution microscopy software.25 P, parasite; R red cell.

Article Snippet: Pan-“PfEMP1” antibodies were raised to recombinant conserved C-terminal acidic-terminal sequence (ATS) domain of PfEMP1 by the commercial vendor Genscript Inc. Anti-PI3P was from Echelon Biosciences.

Techniques: Mutagenesis, Expressing, Sequencing, Infection, Western Blot, Membrane, Staining, Fluorescence, Microscopy, Software