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Blood, Vol. 111, Issue 4, 2418-2426, February 15, 2008
Maurer's clefts of Plasmodium falciparum are secretory organelles that concentrate virulence protein reporters for delivery to the host erythrocyte
Blood Bhattacharjee et al.
111: 2418
Supplemental materials for: Bhattacharjee et al.
Files in this Data Supplement:
- Figure S1: Expression-purification of recombinant HT-GFP and effects of rHT-GFP loaded into cytoplasm of erythrocyte on blood stage infection (PDF, 118 KB) -
(A) Recombinant HT-GFP was expressed in E. coli and purified using Nickel affinity column. Lanes 1 and 2 show proteins from uninduced and induced culture, respectively. Lane 3 shows purified rHT-GFP of 31kDa. (B) Erythrocyte ghosts were loaded with rHT-GFP (top) and invaded with P. falciparum 3D7. Live cell image at the bottom shows ring stage of infection. In all cases p, parasite; ec, erythrocyte cytosol. Bar in fluorescence micrographs (in B) denotes 10mm and 2mm for top and bottom images, respectively. (C) A quantitation for growth characteristic of P. falciparum 3D7 in mock-loaded erythrocyte ghosts (blue) or rHT-GFP- loaded erythrocyte ghosts (brown) compared to non-ghosted human erythrocytes (yellow), measured as a percentage of infected erythrocytes.
- Figure S2. Transgene expression in P. falciparum using piggyBac (PDF, 199 KB) -
(A) Sequence of HT-GFPmembmyc and  -GFPmembmyc with a N-terminal ER-type SS (red) followed by a eleven amino acid HT signal (underlined) contained in a vacuolar translocation sequence (blue) fused to GFP (green), PfEMP1 transmembrane region (pink) and c-myc (orange). (B) Vector diagram of the piggyBac construct pBacII with HT-GFPmembmyc expression cassette placed between cam 5’UTR and hsp86 3’ UTR. Color codes are similar to as described (in A). Filled triangle in -GFPmembmyc signifies replacement of eleven amino acid HT signal (LNKRLLYETQA) with non-specific sequence ISAATDIASTI. IR, inverted repeat sequences; hrp2 3’; histidine rich protein 2 3’ region; dhfr, dihydrofolate reductase; cam 5’ UTR, calmodulin 3’ region; hsp86 3’; heat shock protein 86 3’ region. (C) Southern hybridization of three clones each of HT-GFPmembmyc (left) and -GFPmembmyc (right) indicating the chromosomal integration of piggyBac constructs. Genomic DNA, isolated from respective cloned populations was digested with EcoRV and resolved by agarose gel electrophoresis. Human dhfr probe hybridized to a 12 kb (left, lanes 1-3) fragment in all clones expressing HT-GFPmembmyc and 5 kb fragment in all the three clones expressing -GFPmembmyc (right, lanes 1-3). (D and E) Schematics for the site of integration of piggyBac clones of HT-GFPmembmyc in P. falciparum chromosomes 6 (D) and -GFPmembmyc in chromosome 14 (E).
- Figure S3. Generation of mutant Δctermsbp P. falciparum parasites with a C-terminal deletion in PfSBP1 (PDF, 151 KB) -
(A) Schematic representation for single crossover recombination deleting the C-terminal region of PfSBP1. P. falciparum parasites were transfected with plasmid containing in frame fusion of the neomycin phosphotransferase (npt, green) to an internal fragment of pfsbp1 (orange). Only chromosomal integration of the vector by single crossover with the native pfsbp1 (pink) drives npt expression under the control of pfsbp1 promoter (Ppfsbp1), thus conferring resistance of antibiotic G418. bsd, blasticidin deaminase; cam, calmodulin promoter. (B) PCR-based diagnostic for the loss of chromosomal copy of pfsbp1. Corresponding position of primer pairs used for amplification analyses of single crossover recombination is highlighted (in A). (C) Western blot detecting PfSBP1-NPT fusion product of 70-kDa, by antibodies to NPT (top panel) in transformed line (lane 1) but in not parental line (lane 2). Lower blot, as a loading control, shows the detection of parasite protein PfFKBP of 35-kDa. (D) Indirect immunofluorescence assay of trophozoites expressing C-terminal deletion of PfSBP1 as NPT fusion product. Cells were fixed, permeabilized and probed with antibodies to MSP1 (red, left), NPT (green, middle) and their merge (right). The truncated fusion product is exported to Maurer’s clefts. Parasite nuclei (p) are stained with Hoechst (blue). Bar, 2µ m.
- Figure S4. HT-GFPmembmyc in Δctermsbp, P. falciparum parasites with a C-terminal deletion in PfSBP1 localizes to the Maurer’s clefts (PDF, 47 KB) -
Immunoelectron micrographs of trophozoites expressing HT-GFPmembmyc in ctermsbp parasites. Thin sections (70nm) were probed with antibodies to GFP followed by secondary antibody gold (10nm) conjugate. Arrows indicate gold particles at the Maurer’s clefts (MC). Bar, 200 nm.
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