Figure 1.
Transfection plasmids and detection of GFP fusion proteins.
(A) Plasmid pHH2-KAHRP(+His)-GFP [14] was used to amplify the first 60 amino acids of KAHRP containing the putative hydrophobic signal sequence followed by the histidine rich region with or without GFP fusion. 3′hrp2, histidine-rich protein-2 3′UTR; hDHFR, human dihydrofolate reductase gene; 5′CAM, Pf calmodulin promoter region; 5′hsp86, heat-shock protein-86 promoter region; 3′PcDT, P. chabaudi dihydrofolate reductase 3′UTR; Fp1-KAHRP, forward primer used to add a SpeI restriction site (Sp) immediately before the first codon of KAHRP cloned into pDC-pvcrt-o-MH (not shown); Rp1-GFP-TC, reverse primer used to amplify GFP, add an EcoRV restriction site (Ev) in frame between GFP and the TC tag (for subcloning of other genes), and add a XmaI restriction site (Xm) after the TC sequence, for cloning into pDC-pvcrt-o-MH; Rp2-His-TC, reverse primer used to amplify the histidine rich region of KAHRP, add an EcoRV restriction site (Ev) while maintaining the reading frame of KAHRP(+His) and the TC tag (for future subcloning of other genes), and add a XmaI restriction site (Xm) after the TC sequence, for cloning into pDC-pvcrt-o-MH. (B) Transfection plasmid pDC-KHT contains the sequence for the fusion protein KAHRP(+His)-TC from pHH2-KARHP(+His)-TC. Myc (M) and His×6 (H) tags are encoded immediately after the XmaI restriction site. (C) Transfection plasmid pDC-KHGT contains the sequence of KAHRP(+His)-GFP-TC in frame with codons for a PRGTKTYF terminus (F) that begin at the XmaI site. 5′PcDT, P. chabaudi dihydrofolate reductase promoter region; 3′hsp86, heat-shock protein-86 3′UTR. (D) Immunoblot image shows antibody detection of GFP as a Mr 28,000 band, detection of KAHRP(+His)-GFP-TC as a Mr 42,000 band, and detection of Mr 39,000 and Mr 29,000 bands from KAHRP(+His)-GFP protein. No band was detected from control non-transformed 3D7 PE.
Figure 2.
Fluorescence microscopy images of ReAsH and FlAsH labeled PEs.
(A) Bright field (BF) and fluorescence images of non-transformed 3D7 PE labeled with ReAsH, not treated with BAL. (B) BF and fluorescence images of non- transformed 3D7 PE labeled with FlAsH, not treated with BAL. (C) BF and fluorescence images of 3D7 PE treated with BAL and labeled with ReAsH. (D) BF and fluorescence images of 3D7 PE treated with BAL and labeled with FlAsH. (E) BF and fluorescence images of 3D7-KAHRP(+His)-TC PE treated with BAL and labeled with ReAsH. (F) BF and fluorescence images of 3D7-KAHRP(+His)-TC PE treated with BAL and labeled with FlAsH. (G) BF and fluorescence images of 3D7-KAHRP(+His)-GFP-TC PE treated with BAL and labeled with ReAsH. (H) Images of non- transformed 3D7 PE exposed first to 200 µmol/L CPM and followed by labeling with 2.5 µmol/L ReAsH. Excitation and imaging of ReAsH, FlAsH and CPM fluorescence were performed with wavelengths and filter sets described in Materials and Methods. Scale bar represents 10 µm.
Table 1.
S/N ratios of GFP, FlAsH and ReAsH fluorescence from 3D7-KAHRP(+His)-GFP-TC- and 3D7-KAHRP(+His)-TC-transformed PE.
Table 2.
Measurements of cell multiplication and 50% and 90% inhibitory concentrations (IC50; IC90).
Figure 3.
KAHRP protein trafficking in PEs.
(A–C) Bright field (BF) and GFP fluorescence images of PEs containing Pf 3D7 parasites transformed to express KAHRP(+His)-GFP protein without TC tag. (D–F) Images of 3D7-KAHRP(+His)-GFP-TC PE labeled with ReAsH and photographed in the green and red channels separately for GFP and ReAsH fluorescence. (G–I) Images of 3D7-KAHRP(+His)-TC PE labeled with FlAsH. (J–L) Images of 3D7-KAHRP(+His)-TC PE labeled with ReAsH. The emission crossover between green and red channels was negligible, and was estimated to be less than a 1% leak of GFP emission into the ReAsH emission channel. Scale bar represents 5 µm.
Figure 4.
Photobleaching decay curves of GFP from 3D7-KAHRP(+His)-GFP-TC parasites, and ReAsH and FlAsH from 3D7-KAHRP(+His)-TC labeled parasites in Pf-infected erythrocytes.
Continuous exposure of cells for 20 s to the 9.98 mW 475 nm±20 nm light (for GFP and FlAsH), or to the 10.88 mW 560 nm±22 nm light (for ReAsH) cause fluorescence decays to 65%, 73%, and 85% of the initial emission signals from GFP, FlAsH-, and ReAsH-labeled TC tagged protein, respectively.