2.1. Informatics and phylogenetic tree construction
A family of three paralogous genes, here named the pfm6t
genes, was identified by searching the P. falciparum
genome database for conserved genes encoding one or more transmembrane domains. The encoded PfM6T paralogs were aligned by the Clustal W algorithm using Vector NTI advance 10.1 (Invitrogen, Carlsbad, CA). Transmembrane domain predictions were carried out with TMHMM 2.0 and membrane topology depictions were constructed with TOPO 2.0 (http://www.sacs.ucsf.edu/TOPO-run/wtopo.pl
The 37 orthologous sequences were input into MUSCLE [14
] to generate a multiple sequence alignment. The two M6Tβ fragments for P. chabaudi
(PC000327.01.0 and PC000695.00.0) were combined and assigned to PC000327.01.0, the N-terminal fragment. PY06642 from P. yoelli
appears to be a fragment of PY04760 and was excluded; PB300653.00.0 from P. berghei
was excluded as a fragment of PB000435.02.0. The alignment was improved manually in MacVector 9.5 (MacVector, Cary, NC) and exported as a Nexus formatted file, which was used for a Bayesian phylogenetic analysis in the program MrBayes 3.1 [15
]. The Bayesian phylogeny was the consensus of 7502 post-burn-in samples of two Markov chains each of which ran for 500,000 generations. The tree figure was created using FigTree (http://tree.bio.ed.ac.uk/software/figtree
Peptide fragments were selected based on immunogenicity, synthesized, and KLH-conjugated for antibody production by Spring Valley Labs (Sykesville, MD). Polyclonal antibodies producing specific responses to individual PfM6T proteins were successfully raised in mice (identified with the prefix mp) and rabbits (prefix rp): KFSRYTPYPQDTNQNA-c (rp65α) for PfM6Tα; NVEMGVTENNYIKTAQY-c (rp70β and mp21β) and ARYQQTKSDWTLLHFG-c (rp68β) for PfM6Tβ; ELDIEASTENIAACKQC-c (rp64γ and mp7γ) for PfM6Tγ. A mouse monoclonal antibody for PfM6Tα was also generated using the same synthetic peptide as rp65α.
2.3. SDS-PAGE and Immunoblots
parasite cultures (Indo 1 or W2 isolates) were maintained under standard conditions, enriched by the percoll-sorbitol method [16
], washed, and used for biochemical studies. Membrane and cytosolic fractions were separated by hypotonic lysis (10 mM Na2
, pH 8.0 with 100 µg/mL PMSF, 10 µg/mL leupeptin, 2 µg/mL aprotinin, 2 µM EDTA) and ultracentrifugation at 100,000×g for 1 h at 4 °C. Peripheral proteins were extracted from the membrane pellet in some experiments with 100 mM sodium carbonate, pH 11 [17
]. Protein fractions were separated on 4–12% NuPAGE gels (Invitrogen) under reducing conditions, transferred to nitrocellulose, blocked with 5% powdered milk, and probed with specific antibodies at 1:5000-1:1000 dilutions. A rabbit polyclonal antibody against the SUMO tag of expressed proteins was used at a 1:500 dilution (Life Sensors, Malvern, PA). For detection of immune responses against PfM6T proteins, Ni-NTA purified proteins expressed in insect cells were probed with pooled human serum (1:2000 dilution) from 10 healthy adults living in endemic sites in Mali or Cambodia. These sera were provided by Dr. Rick Fairhurst and were collected with written informed consent under an NIAID IRB-approved protocol. After extensive washing, blots were incubated with HRP-conjugated goat anti-mouse, anti-rabbit, or anti-human IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) at a dilution of 1:10,000 for 1h, washed, and visualized using SuperSignal West Pico substrate (Pierce, Rockford, IL).
For stage specificity analysis of PfM6T expression, in vitro cultures were synchronized with 2 consecutive incubations in 5% sorbitol and cultured for indicated durations before harvesting for immunoblotting of matched samples.
Antibodies (rp65α, rp70β and mp7γ) were separately cross-linked to Dynabeads protein A (Invitrogen) according to the manufacturer's protocol. Infected cell lysate in 0.1M Na2HPO4, pH 8.0 with 1% CHAPS, 100 µg/mL PMSF, 10 µg/mL leupeptin, 2 µg/mL aprotinin was incubated with these beads or with control beads without crosslinking for 30 min before washing extensively and elution by boiling in loading buffer for immunoblot analysis.
2.5. Indirect immunofluorescence confocal microscopy
Indirect immunofluorescence assays (IFA) were performed as described [18
]. Briefly, synchronous cultures of infected erythrocytes were washed to remove serum before making thin smears on glass slides. The cells were air dried, fixed in 1% freshly prepared paraformaldehyde in PBS, washed, and blocked with 5 mg/ml goat serum and 0.1% triton X-100 in PBS. Primary and secondary antibodies were applied in the same buffer at a dilution of 1:100 and incubated at 37 °C for 1hr with extensive washing between antibodies. Colocalization studies used two primary antibodies applied simultaneously. Antibodies against known merozoite surface proteins were mouse monoclonal antibodies against a 42 kD fragment of MSP-1 (R9256/EcMSP-142
) and AMA-1 (4H9/19), both kindly provided by Dr. Sanjay Singh. Species-specific secondary antibodies coupled to fluoropores were used to detect the distribution of these antigens. Goat anti-mouse or anti-rabbit IgGs conjugated to either Alexa Fluor-488 (green) or Alexa Fluor-594 (red) were obtained from Invitrogen. Where shown, nuclear staining was with 300 nM 4, 6-diamidino-2-phenylindole (DAPI). Slides were mounted with Vectashield (Vector Laboratories, Burlingame, CA) and visualized on a Leica SP2 laser scanning confocal microscope (Leica Microsystems, Exton, PA) under a 68x oil immersion objective. Images were processed in Imaris 6.0 (Bitplane AG, Zurich, Switzerland) and uniformly deconvolved using Huygens Essential 3.1 (Scientific Volume Imaging BV, Hilversum, The Netherlands). x-y
plots showing positional fluorescence intensity along the parasite’s apical-posterior axis were also created in Huygens Essential 3.1 and exported to SigmaPlot 10.0 (Systat, San Jose, CA).
2.6. Immunoelectron microscopy
Infected erythrocytes fixed overnight at 4 °C with 0.075% glutaraldehyde/4 % paraformaldehyde were suspended in Hanks buffered saline solution with 10% BSA. 1.5 µl were aliquoted to “hats” (Leica Microsystems, Vienna, Austria) for cryo-immobilization in a Leica EMPact2. Freeze substitution with 1% uranyl acetate/0.1% glutaraldehyde in acetone and dehydration was performed with microwave irradiation (Pelco 3451 microwave processor, 8 cycles of 2 min on–2 min off–2 min on; Ted Pella, Redding, CA) at −78 °C and embedded in LR white resin. Thin sections were cut using an MT-7000 ultramicrotome (Ventana, Tucson, AZ), etched with 4% meta-periodate, and immunolabeled in a Pelco 3451 microwave oven using a Pelco PFTE immunostaining pad. After blocking with 1% BSA/0.1% Tween 20 Tris buffer for 2 min at 150 Watts/24° C (additional steps retained same settings), samples were incubated with primary antibody for 2 × 2 min, washed 3 × 1 min, incubated with secondary 5 nm colloidal gold (BBInternational, Cardiff, UK) for 2 × 2 min before final rinsing. Sections were stained with 1% uranyl acetate and viewed on a Philips CM-10 TEM (FEI, Hillsboro, OR) at 80 kV. Images were acquired with a Hammamatsu XR-100 digital camera system (AMT, Danvers, MA.)
For pre-embed labeling with antibodies, specimens were fixed with 2% paraformaldehyde with 0.0075% glutaraldehyde for 30min, blocked with 10% goat serum for 1 hr, and incubated with rabbit primary antibodies for 1 hr followed by goat anti-rabbit antibody conjugated to 10 nm colloidal gold (BBI International, Cardiff, UK). They were then washed and incubated in 2% paraformaldehyde/0.0075% glutaraldehyde overnight at 4 °C, washed in distilled H2O, dehydrated in ethanol before embedding, sectioning, and staining as above.
2.7. Cloning, baculovirus production, and insect cell expression
The full-length transcript for each paralog was amplified from parasite cDNA with the following primers: GCGGTCTCGAGGTATGTGGTTTACA and GCGAGCTCTTAAGCTGGATAAT (forward and reverse primers, respectively, for PfM6Tα with engineered BsaI and SacI sites underlined), GCGGTCTCGAGGTATGGGTTCAT and GCGAGCTCTCAATATTGAGCTGT (PfM6Tβ with BsaI and SacI sites), GCGGTCTCGAGGTATGTTTTTTACTTA and GCGGATCCTTAACATTGTTTGCA (PfM6Tγ with BsaI and BamHI sites). Each amplicon was digested with BsaI to produce a sense strand 5’ overhang complimentary to an ACCT antisense strand overhang in the iCTHS vector (Life Sensors, Malvern, PA, USA). Ligation into this vector then created a single open reading frame encoding a 6-histidine tag (6-His) followed by a half-SUMO tag (CTHS) immediately upstream of the pfm6t gene. After cloning and confirming sequence, each plasmid was recombined into an expression bacmid as described (Bac-to-Bac baculovirus expression system, Invitrogen, Carlbad, CA, USA). Successful transformation was selected with 50 µg/mL kanamycin, 7 µg/mL gentamicin, 10 µg/mL tetracycline, and 100 µg/mL Bluo-gal (Technova, Hollister, CA, USA); recombinants were selected with blue-white screening using IPTG, sequenced to exclude mutations, and subjected to bacmid harvest for transfection of Spodoptera frugiperda (Sf9) insect cells with Cellfectin (Invitrogen). After a 72 h incubation at 28 °C, baculovirus released into the supernatant was harvested, used to confirm the gene’s presence by PCR, and passaged further in Sf9 or Trichopulsia ni (High Five cells, Invitrogen). Viral titers were determined by plaque assays with Neutral Red (Invitrogen). Expressed protein was harvested from suspension cultures using recombinant baculovirus at a multiplicity of infection (MOI) of 10 and a 72 h incubation at 28 °C.
Indirect immunofluorescence confocal microscopy was performed using a sterile chamber with a coverglass bottom with adherent insect cells transfected with baculovirus at a MOI of 3 and fixation in 1% paraformaldehyde before labeling.
2.8. Ni2+-NTA purification of expressed protein
Transfected T. ni cells were suspended in 25 volumes of 50 mM tris-HCl (pH 7.5) with 250 mM sucrose, 20 mM β-mercaptoethanol, 100 µg/mL PMSF, 10 µg/mL leupeptin, 2 µg/mL aprotinin and 20 µg/mL DNase, and subjected to 3 freeze-thaw cycles with sonication. Membranes were then isolated by ultracentrifugation (100,000 × g, 1 h at 4 °C) before solubilization in 50 mM Na2HPO4 buffer at pH 7.0 containing 8 M urea, 1 % CHAPS, 1 % n-dodecyl-β-D-maltoside and 1 % n-octyl-β-D-glucoside with a dounce homogenizer. After removing insoluble material by ultracentrifugation, solubilized proteins were subjected to Ni2+-NTA purification using the ProBond Purification System (Invitrogen). 6-His tagged proteins were allowed to bind to the column under continuous agitation for 12 h, washed with 10 mM imidazole, pH 6.0 in the above buffer, and eluted in the buffer with 200 mM imidazole, pH 4.0.
2.9. Reconstitution into liposomes and SUMO protease digestion
Soybean azolectin (lecithin, type II; Sigma Aldrich, St. Louis, MO) was sonicated at 4 mg lipid /ml in 40 mM tris-HCl (pH 7.5) with 4 mM DTT and 0.2 mM EDTA to homogenity. Ni2+-NTA purified protein reconstituted into liposomes by mixing with this lipid and extensive dialysis against 50 mM Na2HPO4, pH 7.0 with methanol-washed SM2 Bio-Beads (Bio-Rad, Hercules, CA). A 50 µl liposome suspension in PBS was incubated with 20 µg N-terminal half SUMO (NTHS, Life Sensors) for 30 min at 30 °C with intermittent sonication before addition of 40 units SUMO protease (Life Sensors) with 2 mM DTT. The mixture was incubated overnight at 4 °C to permit specific cleavage of the chimeric protein. This technology is designed to prevent CTHS cleavage by endogenous SUMO proteases in insect cells, but allow cleavage by exogenous SUMO protease after protein purification because association of NTHS with CTHS restores protease sensitivity. This cleavage yields unmodified, full length PfM6T protein without the 6-His-CTHS tag.