Intramembrane proteolysis mediates shedding of a key adhesin during erythrocyte invasion by the malaria parasite

Abstract

Apicomplexan pathogens are obligate intracellular parasites. To enter cells, they must bind with high affinity to host cell receptors and then uncouple these interactions to complete invasion. Merozoites of Plasmodium falciparum, the parasite responsible for the most dangerous form of malaria, invade erythrocytes using a family of adhesins called Duffy binding ligand-erythrocyte binding proteins (DBL-EBPs). The best-characterized P. falciparum DBL-EBP is erythrocyte binding antigen 175 (EBA-175), which binds erythrocyte surface glycophorin A. We report that EBA-175 is shed from the merozoite at around the point of invasion. Shedding occurs by proteolytic cleavage within the transmembrane domain (TMD) at a site that is conserved across the DBL-EBP family. We show that EBA-175 is cleaved by PfROM4, a rhomboid protease that localizes to the merozoite plasma membrane, but not by other rhomboids tested. Mutations within the EBA-175 TMD that abolish cleavage by PfROM4 prevent parasite growth. Our results identify a crucial role for intramembrane proteolysis in the life cycle of this pathogen.

Figure 1.

EBA-175 is shed at or around the point of erythrocyte invasion and retains region VI. (A) Schematic of EBA-175 structure, indicating signal sequence (SS), F1 and F2 DBL domains, TMD, and cytoplasmic domain (CYT). The position of region VI is shown, with a Coomassie blue–stained SDS-PAGE gel of purified recombinant region VI (inset). (B) Western blot of 3D7 schizonts, merozoites, and culture supernatant, run on nonreducing 7.5% SDS-PAGE and probed with anti–region VI antibodies. Shed EBA-175 (arrow) migrates more rapidly than the parasite forms. Molecular mass markers are indicated. (C) IFA images of an acetone-fixed segmented 3D7 schizont and free merozoites dual labeled after fixation with mAb 1E1 (anti-MSP1₁₉; red) and anti–region VI antibodies (green). The punctate, apical pattern obtained with the latter is typical of microneme staining. (D) EBA-175 is discharged onto the apical surface of free merozoites. Merozoites released from 3D7 schizonts in the presence of anti–region VI antibodies were washed, fixed, and probed with FITC anti-mouse IgG to detect bound anti–region VI antibodies (green), followed by mAb 1E1 (anti-MSP1₁₉; red). Many free merozoites exhibit a strong apical FITC signal (thin arrows), whereas residual intact schizonts containing merozoites that were not accessible to the anti–region VI antibodies show only background FITC labeling (thick arrows). No signal was associated with merozoites released in the presence of preimmune mouse antisera (unpublished data). (E) IFA images of newly invaded (≤3-h-old) ring-stage 3D7 parasites probed with anti–region VI antibodies after acetone fixation. Most rings did not react at all with the antibodies. Nuclei were stained with DAPI (blue). Identical results were obtained with the W2mef clone (unpublished data). Bars, 5 μm.

Figure 2.

EBA-175 is not shed by PfSUB2 but by a distinct calcium-independent serine protease. Shed EBA-175 and processing fragments of AMA1 (AMA1_48/44) and MSP1 (MSP1₃₃) were detected in merozoite supernatants by Western blot as described in Materials and methods. Supernatants were harvested immediately (start) or after a 1-h incubation at 37°C in buffer alone (no additions) or containing the indicated additives. Isopropanol and DMSO were solvent controls for PMSF and dichloroisocoumarin (DCI), respectively, whereas FT control refers to flow-through from the final step of concentration of purified PfSUB2PD by ultrafiltration (Harris et al., 2005), added to the assay at a dilution similar to that used for PfSUB2PD. These results were reproducible in three independent experiments. In Western blot analysis (similar to that in Fig. 1 A) the shed EBA-175 produced in these assays comigrated with that from culture supernatants (unpublished data), consistent with it being the result of the same protease activity.

Figure 3.

EBA-175 is shed by intramembrane cleavage. (A) Coomassie-stained SDS-PAGE of partially purified shed EBA-175 (arrow). A total of ∼40 μg of EBA-175 was obtained from 3 liters of culture medium. (B) The most C-terminal intact tryptic and Asp-N peptides identified in digests of EBA-175, with calculated m/z values in carbamidomethylated form, are shown in relation to the juxtamembrane sequence of 3D7 EBA-175 (PlasmoDB ID PF07_0128). The TMD (TMHMM v2.0) is shaded. The sequence is aligned with corresponding sequence from P. falciparum paralogues EBA-181 (PFA0125c) and EBA-140 (MAL13P1.60), as well as P. falciparum MAEBL (PF11_0486), P. vivax DBP (available from GenBank/EMBL/DDBJ under accession no. P22290), and P. knowlesi DBP (available from GenBank/EMBL/DDBJ under accession no. P22545). Conserved cysteines are marked with an asterisk. Helix breaking motifs constituting potential rhomboid recognition sites within the TMD are underlined, and the conserved Ala at which EBA-175 is cleaved is boxed. (C) MALDI-TOF spectra of EBA-175 Asp-N digests performed in 100% H₂ ¹⁶O or 50% (vol/vol) H₂ ¹⁸O. The peak matching ¹⁴⁰⁸DDPSYTCFRKEAFSSMPYYA¹⁴²⁷ (arrow; shown magnified in inset, with observed m/z value) is not ¹⁸O-labeled in the lower spectrum, indicating that it derives from the C terminus. For comparison, peaks corresponding to ¹²²⁷DRNSNTLHLK¹²³⁶ (calculated m/z 1197.634) and ⁵⁶²DLSNRKLVGKINTNSNYVHRNKQN⁵⁸⁵ (calculated m/z 2812.493) are indicated as examples of species that in the lower spectrum exhibit the ¹⁸O-labeled isotope pattern typical of internal peptides. (D) Collision-induced fragmentation spectrum of the unlabeled m/z 2435.242 peptide in Asp-N digests. The indicated fragment peaks can be assigned to a b and y series of ions, with the b₁₈ and b₁₉ ions assigning the C-terminal peptide sequence Y-A. The remaining major ions are all consistent with the indicated sequence.

Figure 4.

PfROM4 is a merozoite plasma membrane protein. (A) Western blot showing extracts of parental 3D7 and 3D7HAROM4_synth probed with mAb 3F10 (anti-HA) and polyclonal anti-AMA1 (loading control). The size of the anti-HA–reactive species detected only in 3D7HAROM4_synth is close to the predicted mass (90.4 kD) of HA-tagged PfROM4. (B) IFA of mature nonsegmented (top) and fully segmented (bottom) schizonts of 3D7HAROM4_synth, dual labeled with mAb 3F10 (anti-HA; green) and mAb 1E1 (anti-MSP1; red). The DAPI signal (blue) is not included in merged images for clarity. Bar, 5 μm.

Figure 5.

EBA-175 is a substrate for PfROM4. (A) Structure of EBA-175 minigenes expressed in COS-7 cells. Partial amino acid sequences of the various mutants are indicated. Substitutions made to remove N-glycosylation sites or to modify the TMD (shaded) are in bold and underlined. The position mapped as the site of EBA-175 shedding is indicated by an arrow. The predicted topology of the expression products is shown (inset). (B) IFA showing expression of EBA-175 minigene products at the surface of COS-7 cells. Cells transfected with the various constructs were probed without fixation or permeabilization with the anti-HA mAb 3F10 (green). In all cases, 15–20% of cells exhibited strong surface fluorescence. Nuclei were stained with DAPI (blue). Bar, 50 μm. (C) Western blots of cells or medium from COS-7 cells cotransfected with EBA-175 minigene constructs and HA-tagged PfROM4 expression constructs, either in wild-type (wt) or active site Ser knockout form (mut). Blots were probed with mAb 3F10. The uppermost band visible in the cell extract blot (arrow) corresponds to HA-tagged PfROM4; note its absence from the farthest right lane, where no PfROM4 construct was transfected. The band just below this is a nonspecific reaction signal. These results were reproducible in >19 independent experiments.

Figure 6.

Mutation of the PfROM4 recognition motif within the EBA-175 TMD is deleterious. (A) Scheme showing the replacement of the 3′ region of the eba-175 gene by single-crossover integration of pHH1-175-GA_1,2/FF constructs. The positive selection cassette (hDHFR) of the pHH1 vector is represented by a black box. A ∼1-kb fragment of eba-175 cDNA including sequence encoding region VI (yellow), the mutant TMD (red), and the cytoplasmic domain (green) was cloned into vector pHH1, flanked by the 3′ UTR of the Plasmodium berghei dihydrofolate reductase gene (blue) to ensure correct transcription termination and polyadenylation of the modified gene. The intron-exon structure of the eba-175 gene is shown (gray; signal peptide). MfeI restriction enzyme sites are indicated (M). Relative positions of oligonucleotides used in the PCR analysis (1, primer HH1-S; 2, primer HH1-AS; 3, primer 175₃₂₅₉-S; 4, primer 175-AS_stop; see Table S2, available at http://www.jcb.org/cgi/content/full/jcb.200604136/DC1) are shown as colored arrows, and the position of the probe used for Southern analysis is indicated by the red bar. (B) Southern blot of gDNA from parental and transgenic parasites (MfeI restricted) reveals integration of pHH1-175-GA₂/FF but not pHH1-175-GA₁/FF into the eba-175 locus. The endogenous eba-175 hybridizing fragment (8.4 kb) is present in the W2mef and 3D7 EBA175GA₁/FF lines at both the second and third cycles of drug selection but disappears upon integration of the plasmid in the W2mef EBA175GA₂/FF transgenic line; the 2.4- and 13-kb hybridization bands indicate replacement as expected of the 3′ end of the eba-175 gene by the second drug selection cycle. Sizes of hybridizing bands are shown, and the band corresponding to free episome is marked by an arrow. (C) PCR analysis of the eba-175 locus using gDNA from parental and transgenic parasite lines. Molecular mass markers are shown (M). Amplification from the eba-175 locus is displayed for each parasite line using three different oligonucleotide primer combinations: 1 plus 2 (black and blue; plasmid specific), 3 plus 4 (red and green; eba-175 specific), and 2 plus 3 (blue and red; eba-175 single-crossover specific). (D) IFA localization of mutant EBA-175 in transgenic EBA175GA₂/FF W2mef schizonts and free merozoites. Antibodies specific for EBA-175 (red) and EBA-181 (green) colocalize in the merged image. The DAPI signal (blue) is not included in the merge. Bars, 5 μm.