Fig 1.
PTP7 localizes to the Maurer’s clefts and other compartments in the host cell.
(A) Schematic of PTP7. Amino acid numbers are indicated. SS: Signal sequence. KSL/AE: export element; TM: transmembrane domain; 34xN: 34 consecutive asparagine amino acids. (B) Live cell microscopy of PTP7-GFPsand infected RBCs showing native fluorescence (green) merged with bright field (BF). (C) Indirect immunofluorescence microscopy of paraformaldehyde-fixed PTP7-GFPsand infected RBCs synchronized to a 2-hour window and sampled every 4 hours from 16 to 28 hours post invasion (hpi). The infected RBCs were probed with anti-GFP (green), anti-REX1 (magenta), and DNA was stained with DAPI (blue). (D) Percentage of Maurer’s clefts labeled with both anti-GFP and anti-REX1, by object segmentation. Data displayed are mean ± SD of the means per biological repeat. (E) Super-resolution microscopy of indirect immunofluorescence assays of infected RBCs synchronized to 24–26 hpi probed with the antibodies indicated. (F) Representative immuno-transmission electron microscopy (TEM) micrographs of 20–32 hpi infected RBCs permeabilized with Equinatoxin-II. Cells were probed for FKBP followed by immunogold secondary labeling. V: vesicles, MC: Maurer’s cleft, RBCM: red blood cell membrane. White arrows highlight gold labelling.
Fig 2.
Disruption of the PTP7 locus affects knob and cleft morphology.
(A) Schematic outlining the gene disruption strategy. DSB: double stranded break; RNP: ribonucleoprotein; yDHODH: yeast dihydroorotate dehydrogenase; Gray: coding sequence; up-caret: native intron; blue bulb and purple line: RNP and small guide RNA; HR1: homology region 1; HR2: homology region 2; crossing lines: homologous cross over events; arrows and letters A-D: primer locations. (B) PCR products of CS2 and CS2ΔPTP7 genomic DNA confirming disruption of the ptp7 locus. (C) Immunoblot of cell lysates probed with αPTP7. Loading control αBiP, expected size of ~62 kDa. (D-E) Indirect immunofluorescence microscopy of acetone/methanol fixed cells. Bright field (BF) and DAPI stained DNA (blue) images are merged. Scale bar, 2 μm. (F) Scanning electron microscopy of the exterior surface of mid-trophozoite stage infected RBCs. (G) Knob density as knob count per square μm, averaged per image. Data displayed are mean ± SD (n = 13, 10). (H) Data points represent the average knob diameter along the major axis for knobs in an individual cell. Data displayed are the mean ± SD (CS2 n = 13; ΔPTP7 n = 10). (I) Electron tomograms of infected RBCs membranes reveal the spiral structure underlying the knob. P-values determined by Welch’s t-test, n values are individual cells from ≥ 2 biological repeats.
Fig 3.
PTP7 disruption affects EMP1 distribution and surface presentation.
(A) Infected RBCs were analyzed for their adherence to chondroitin sulfate A (CSA). Infected CS2 and CS2ΔPTP7 cell lines were passed through a channel slide coated with CSA under physiological flow conditions. The number of infected RBCs in 10 fields of view were recorded. Samples were run in technical triplicate and the experiment was repeated 3 times. Data displayed are mean ± SD of each technical repeat (CS2 n = 3; ΔPTP7 n = 3). (B) Flow cytometry analysis of infected RBCs labeled with antibodies recognizing the ectodomain of var2CSA, followed by secondary antibodies and tertiary antibodies conjugated to Alexa Fluor 488. Samples were run in triplicate and the experiment was repeated 3 times. The relative mean fluorescence of events was calculated as the (FITC geometric mean × number of events × 105) and averaged per cell line for each biological repeat. Data displayed are mean ± SD of each technical repeat (CS2 n = 3; ΔPTP7 n = 3, symbols indicate different biological repeats). (C) Trypsin cleavage assay. Membranes are probed with αATS and αSBP1 as a control. P: PBS mock treatment; T: Trypsin treated samples; asterisk: spectrin cross-reactivity band; arrows: trypsin cleaved EMP1 products. Loading control and experimental control, αSBP1, expected molecular weight of ~50 kDa [28]. (D) Indirect immunofluorescence assays of cells fixed in acetone/methanol then probed with antibodies to the acidic terminal segment (ATS, green) and the Maurer’s cleft protein REX1 (magenta) and stained for DNA using DAPI (blue). Example merged (left) and mask (right) images are shown, where EMP1 only (green), REX1 only (magenta) and both (white) objects are distinguished. White arrows indicate structures containing both REX1 and EMP1 (ATS). Scale bar, 2 μm. (E) Quantitation of the percent of EMP1 positive REX1 labeled structures. Data displayed are mean ± SD (CS2 n = 44; ΔPTP7 n = 63). (F) A representative example of the cleft and RBC cytoplasm masks used to quantify fluorescence intensity. The same cell as shown in D is represented here. (G, H) Quantification of the number and size of the Maurer’s clefts in the CS2 and ΔPTP7 infected RBCs. Data displayed are mean ± SD (CS2 n = 41; ΔPTP7 n = 68). P-values determined by Welch’s t-test, n values are individual cells from ≥ 2 biological repeats.
Fig 4.
PTP7 disruption leads to an accumulation of vesicles at the clefts.
(A-B) Immuno-electron micrographs of 20–32 hpi CS2 and ΔPTP7 infected RBCs permeabilized with Equinatoxin-II and probed for REX1 (A) (αREX1-repeats [63]) or EMP1 (B) (αR3031 [40]) followed by immunogold secondary labeling with protein-A (EM grade, 6 nm gold). K: knob, V: vesicle, MC: Maurer’s cleft, RBCM: red blood cell membrane. (B) Immuno-electron micrographs of αR3031 (EMP1) labeled cells. Magenta arrows: gold labeled puncta, white arrows: gold labeled vesicles. (C) Clefts modeled from tomogram reconstructions of each cell line. Maurer’s clefts, green and blue hues; tethers, white stalks. Scale bar, 200 nm. Translations through the tomograms and rotations of the rendered models in S1–S4 Movies.
Fig 5.
PTP7 C-terminus is required for wildtype knob morphology.
(A) Schematic of the PTP7 primary sequence. Numbers indicate amino acid position. SS: Signal sequence; TM: transmembrane domain; N mono-repeats: asparagine repeats. (B) Immunoblot of lysates of the parent line (CS2) and the PTP7 C-terminally truncated lines probed with αGFP. Expected sizes of GFP chimeras from left to right are 58, 51, 54, and 56 kDa. Loading control, αGAPDH, expected size of ~38 kDa. (C) Mid-trophozoite stage infected RBCs were fixed in 2.5% glutaraldehyde/PBS and prepared for SEM of the exterior surface. Scale bar indicated. (D-E) Quantitation of the (D) knob density as knob count per square μm and (E) mean knob diameter in nm. Data displayed are mean ± SD (n = 10 per cell line). P-values determined by Brown-Forsythe and Welch ANOVA tests and Dunnett’s multiple comparison test, n values are individual cells.
Fig 6.
A full-length C-terminus is required for antigen delivery and wildtype cleft morphology.
(A) Flow cytometry analysis of infected RBCs labeled with antibodies to the ectodomain of var2CSA followed by secondary antibodies and tertiary antibodies conjugated to Alexa Fluor 647. Samples were run in technical duplicates. Data displayed are mean fluorescence values ± SD for each biological repeat. 4 biological repeats are displayed, each with their own shape. (B) Trypsin cleavage assay of truncation lines. Membranes were probed with αATS and the loading/experimental control αSBP1. P: PBS mock treatment; T: Trypsin treated samples; asterisk: spectrin cross-reactivity band; arrows: Trypsin cleaved EMP1 products. Loading control and experimental control, αSBP1 expected molecular weight is ~50 kDa. (C) Equinatoxin-II permeabilized infected RBCs probed with αGFP followed by immunogold secondary labeling. (D-E) Quantitation of vesicles from immuno-electron micrographs. (D) Mean vesicle diameter per image. Data displayed are mean ± SD (n = 32, 41, 24, 24 respectively). (E) Number of vesicles within 100 nm of each cleft. Data displayed are mean ± SD (n = 43, 73, 38, 42 respectively). P-values determined by Brown-Forsythe and Welch ANOVA tests and Dunnett’s multiple comparison test, n values are individual cells.