Exported Proteins Required for Virulence and Rigidity of Plasmodium falciparum-Infected Human Erythrocytes

Summary A major part of virulence for Plasmodium falciparum malaria infection, the most lethal parasitic disease of humans, results from increased rigidity and adhesiveness of infected host red cells. These changes are caused by parasite proteins exported to the erythrocyte using novel trafficking machinery assembled in the host cell. To understand these unique modifications, we used a large-scale gene knockout strategy combined with functional screens to identify proteins exported into parasite-infected erythrocytes and involved in remodeling these cells. Eight genes were identified encoding proteins required for export of the parasite adhesin PfEMP1 and assembly of knobs that function as physical platforms to anchor the adhesin. Additionally, we show that multiple proteins play a role in generating increased rigidity of infected erythrocytes. Collectively these proteins function as a pathogen secretion system, similar to bacteria and may provide targets for antivirulence based therapies to a disease responsible for millions of deaths annually.


Culture conditions and parasite strain.
Erythrocytic stages of P. falciparum were maintained in human 0+ erythrocytes (Crabb et al., 1997). CS2 wild-type parasites, a clone of the It isolate (Rogerson et al., 1995), adheres to chondroitin sulphate A (CSA) and hyaluronic acid in vitro. Parasites were selected for the adherence to bovine trachea CSA (Sigma, ST Louis, MO, USA) prior to transfection.

Plasmid constructs, transfection and Southern blotting.
Constructs were either assembled in pHHT-TK (Duraisingh et al., 2002) or pCC1 (Maier et al., 2006) vectors (see below). The vectors contain a hDHFR cassette (driven by a calmodulin promoter) flanked by 2 multiple cloning sites to accept targeting sequences of the relevant gene. They also include a negative selection cassette (driven by the Hsp86 promoter region) to select parasites in which double recombination events had occurred. Plasmid DNA was extracted using Maxiprep kits from either Qiagen or Invitrogen (Purelink). 80 µg DNA was tranfected for each transgenic line using standard protocols (Crabb et al., 1997). After positive selection on WR99210 the cells were placed under negative selection using either Ganciclovir (Roche, 20µM, pHHT-TK) or 5-Fluorocytosine (ICN, 100 nM, pCC1). If no cells were recovered the negative selection was repeated at least twice. Any resulting cell populations underwent Southern blot analysis. Genomic DNA was prepared with the Dneasy Tissue Kit (Qiagen) and Southern Blot analysis performed using the DIG system (Roche) according to manufacturer's instructions to confirm disruption of the targeted genes. In assembling pCC1 the goal was to construct a modular vector with gene cassettes for negative and positive selection and multiple cloning sites for the incorporation of gene-specific targeting sequences. We modified pGEM7Z(+) (Promega) by annealing a polylinker consisting of oligonucleotides aw132 and aw133 into the XbaI/SacI sites of pGEM7Z(+). This yielded the cloning vector LT-1. We then amplified firefly luciferase from pPf86 (kindly provided by Kevin Militello, Harvard School of Public Health) with the primer pair aw118/119 and cloned it into the BamH I/Hind III sites of LT-1 creating the vector LT-2. The 3' UTR of the gene encoding the histidine rich protein 2 (HRP2 3') was cut out of the vector pHHT-TK (Duraisingh et al., 2002) with Hind III/EcoR I and annealed into LT-2. The 5' UTR of the P. falciparum calmodulin (CAM) gene was amplified with the primer pair aw76/77 from pHHT-TK and ligated into the HRP2 3' containing LT-2. This plasmid was named LT-3.
The 3' UTR of the P.berghei dihydrofolate reductase/thymidylate synthase (PbDT 3') was amplified with the oligonucleotides aw122/123 and ligated into the Not I/Xma I cut plasmid pHHT-TK resulting in pHHT-TK-3'. The firefly luciferase was cut out of LT-3 with BamH I/Hind III and replaced with the human dihydrofolate reductase gene (hDHFR) from pHHT-TK. The hDHFR containing LT-3 was then cut with EcoR I/Afl II to release the whole hDHFR gene cassette (with the CAM5' and HRP2 3') and cloned into EcoR I/Afl II cut pHHT-TK-3'.
The resulting vector was named pDC1 and contains a CAM5'-hDHFR-HRP2 3' gene cassette for positive selection and a HSP86 5'-Herpex simplex TK-PbDT 3' gene cassette for negative selection. The component of each gene cassette can be individually cut out and replaced (hence the vector is modular). Each gene cassette is flanked by a multiple cloning site. In addition the vector contains a plasmid backbone, which enables ampicillin selection in E. coli and replication both in E.coli and P.falciparum. Finally the ScCDUP gene was amplified with the primers aw500 and aw 501 from the plasmid pHHT-CDUP-ΔPF11_0037 (Maier et al., 2006) and cloned Xho I/Xma I into the cut pDC-1 to replace the HsTK gene with the ScCDUP gene. The final vector was then called pCC-1.

Generation of antibodies.
The following KLH-coupled fusion peptides were synthesised (Invitrogen) and immunized. IgG from the rabbit sera were purified via a protein G sepharose column and eluted with 100 mM glycine-HCl, pH 2.5, dialysed against PBS and their concentration adjusted to ~5 mg/ml.

CSA adherence assays.
Static binding assays (50 mg/ml) and binding under physiological flow conditions (100 mg/ml) to CSA were performed using P. falciparum-infected erythrocytes at 3% parasitemia and 1% hematocrit (Crabb et al., 1997). For panning purposes plastic petri dishes were coated with 100 µg/ml CSA overnight and blocked with 10% human serum in RPMI-HEPES. Synchronised cultures at the trophozoite stage were enriched via gelatine flotation and added to the CSA coated petri dishes, where adhesion was allowed to occur for 1 h at 37 o C. Unbound cells were washed off with three washes with RPMI-HEPES, pH6.8. Bound cells were resuspended and taken into culture to expand, before another round of selection commenced. After 3 rounds of selection the cultures were analysed for the expression of PfEMP1 via a trypsin cleavage assay.

Flow based cytoadherence assays.
Flow assays on protein-coated microslides were performed using standard conditions (Tse et al., 2004), with a coating concentration of CSA of 100 µg/ml. All cell lines were tested in duplicate in three separate experiments. The results are expressed as number of bound infected red blood cells per mm 2 . Cell lines displaying binding values outside of the 95% confidence interval of the CS2 parental line were regarded as having a different binding phenotype.

Trypsin cleavage assays.
For trypsin cleavage sorbitol synchronised parasites were grown to trophozoite stage and enriched via gelatine [Gelofusine, Braun, Bella Vista, Australia] flotation.
Infected red blood cells were then either incubated in TPCK-treated trypsin (Sigma) (1 mg/ml in PBS), in PBS alone or in trypsin plus soybean trypsin inhibitor (5mg/ml in PBS, Worthington, Lakewood, NJ, USA) at 37 o C for 1h. Trypsin inhibitor was then added to the trypsin and PBS aliquot to be incubated at room temperature for 10 min (Waterkeyn et al., 2000). Cell pellets were extracted in the presence of protease inhibitors (Complete, Roche) with Triton X-100 (1%) and subsequently with sodium dodecylsulfate (SDS, 2%) as previously described (Baruch et al., 1996).

Laser-assisted optical rotational cell analysis.
To measure deformability the infected red blood cells were subjected to analysis via a laser-assisted optical rotational cell analyser (LORCA). In this assay erythrocytes are taken up in a polymer solution sitting in a gap between an inner cylinder and an outer cup. By rotating the cup shear stress is created, which in turn forces the erythrocytes to change from a biconcave to an ellipsoid morphology.
Samples were analysed using a FACSCalibur flow cytometer (Becton-Dickinson, USA) and Flowjo software (TreeStar, USA). Fluorescence in channel FL1 was used as a measure of IgG binding and for each sample the geometric mean fluorescence of uninfected red blood cells was deducted from the geometric mean fluorescence of infected erythrocytes. All samples were tested in duplicate.

Serum samples.
Sera were collected from malaria-exposed pregnant residents of the Madang Province, Papua New Guinea (PNG), presenting for routine antenatal care at the Modilon Hospital, Madang. This population experiences year-round transmission of P.
falciparum. Sera from non-malaria exposed Australian residents were included as controls. Written informed consent was given by all donors and ethical clearance was obtained from the Medical Research Advisory Committee, Department of Health, PNG, and the Walter and Eliza Hall Institute Ethics Committee.

Southern blot analysis for all obtained gene knock-outs.
Genomic DNA from CS2 and transfected cell lines was digested with indicated combinations of restriction enzymes and hybridised with the 5' or 3' targeting region of the deleted gene. Expected sizes for wild-type (WT) locus (3D7 strain), for the locus with integration of the hDHFR cassette via double recombination and for the plasmid are indicated in kilobases (kb).  To ensure that the reduced binding of erythrocytes infected with the transgenic lines CS2∆PFA0620c, CS2∆PFB0090c and CS2∆PFE0060w is due to a switch to the expression of another var gene these cultures were subjected to "panning" on CSA.
After 3 rounds of selection these cell lines were referred to as PFA0620c up, PFB0090c up and PFE0060w up, respectively, and a trypsin cleavage assay was performed. The full-length PfEMP1 and the cytoplasmic tail were detected using antibodies to the acidic terminal segment (ATS) at the C-terminus of PfEMP1. The lanes for each parasite-infected red blood cell show: untreated (-), trypsin treated (+) and treated with trypsin and soybean trypsin inhibitor (i). As a comparison CS2 wildtype infected erythrocytes (CS2) (which express var2CSA PfEMP1) and uninfected red blood cells were subjected to trypsin cleavage too. Full-length var2CSA PfEMP1 and the two trypsin-resistant bands at 70 and 90 kDa are indicated by arrows. After 3 rounds of CSA panning the majority of cells had been selected for the expression of var2CSA, although (especially in erythrocytes infected with PFA0620c up and PFE0060w up) there was still a detectable subpopulation expressing another PfEMP1 as indicated by additional trypsin-resistant bands at 80-90kDa and additional full length bands of different sizes. However, these experiment show that the deletion of these genes are neither responsible for the switch in the PfEMP1 nor that this prevents the cells from reverting to the expression of var2CSA. In addition it shows that in these cells -independent from the var gene expressed -PfEMP1 is still exported to the surface of the infected red blood cell. Functionally erythrocytes infected with these CSA up-selected parasite lines display an increased ability to bind to CSA (Fig.  3C) and are being increasingly recognised by var2CSA specific antibodies by FACS assays (Fig. 3A).  The first column in each panel shows a bright field image, the second the DAPI nuclear stain, the third the PfEMP3 (Waterkeyn et al., 2000) or SBP1 (Cooke et al., 2006;Maier et al., 2007) fluorescence and the fourth an overlay of the previous images. No major differences were observed in these cell lines. One representative cell of >30 examined is shown. Figure S7.

Immunofluorescence analysis of all mutant cell lines generated.
Mutants were screened for defects in PfEMP1, PfEMP3, KAHRP and SBP1 trafficking with no major differences observed. The first column in each panel shows a bright field image, the second the DAPI nuclear stain, the third PfEMP1 (Maier et al., 2007), KAHRP (Rug et al., 2006), PfEMP3 (Waterkeyn et al., 2000) or SBP1 (Cooke et al., 2006;Maier et al., 2007) fluorescence, respectively, and the fourth an overlay of the previous images.