Erythrocyte-binding antigen 175 mediates invasion in Plasmodium falciparum utilizing sialic acid-dependent and -independent pathways

Abstract

The Plasmodium falciparum erythrocyte-binding antigen 175 (EBA-175) is a ligand for merozoite invasion into human erythrocytes that binds to glycophorin A in a sialic acid-dependent manner. P. falciparum strain W2mef depends on sialic acid for invasion of erythrocytes, whereas 3D7 is sialic acid-independent. We generated parasites that lack expression or express truncated forms of EBA-175 in W2mef and 3D7. Lack of EBA-175 expression in W2mef parasites was associated with a switch to sialic acid-independent invasion. 3D7 parasites lacking expression of EBA-175 showed no alteration in their ability to utilize sialic acid-independent pathways. Strikingly, both W2mef and 3D7 parasites lacking EBA-175 expression invaded chymotrypsin-treated erythrocytes inefficiently compared with the parental lines. This loss of function suggests that the EBA-175/glycophorin A ligand-receptor interaction is the major chymotrypsin-resistant invasion pathway. Parasite lines with truncated EBA-175 had invasion phenotypes equivalent to parasites lacking expression of EBA-175. The EBA-175 ligand is functional in erythrocyte invasion by merozoites that utilize either sialic acid-dependent or -independent invasion pathways. This finding suggests a model where a minimal affinity supplied by multiple ligand-receptor interactions is required for successful invasion and has implications for EBA-175 as a malaria vaccine candidate.

Figure 1

Disruption of EBA-175 in two lines of P. falciparum that invade by either sialic acid-dependent or -independent pathways. (A) Structure of EBA-175. Shown are signal sequence, F1 and F2 domains that bind to glycophorin A, 3′ cysteine-rich region, transmembrane domain, and cytoplasmic domain. The black boxes above the protein refer to regions where crossover occurred in the gene sequence. This crossover resulted in deletion of the F1/F2 region from the genome. (B) Disruption of the EBA-175 gene in 3D7 and W2mef. The pHTkΔ175 plasmid contains the thymidine kinase gene (Tk), hDHFR, and 5′ and 3′ EBA-175 regions. EBA-175 is shown with homologous target sequences in black (5′ flank) and gray (3′ flank). The double-crossover integration events are shown for both 3D7Δ175 and W2mefΔ175 where the F1/F2 region has been deleted. The single-crossover recombination events for 3D7Δ175/3′ and W2mefΔ175/3′ are shown where one copy of the full transfection plasmid has integrated. Sizes of DNA fragments are shown in kb. Restriction enzyme sites are N, NsiI; Ba, BanI; E, EcoRV; and Bg, BglII. PrA and PrB are hybridization probes used in C–E. (C) Southern blot of genomic DNA from parasites shown digested with NsiI or BanI/EcoRV and probed with PrA. (D) Southern blot of genomic DNA from the parasites shown digested with NsiI or BanI/EcoRV and probed with PrA. (E) Southern blot of genomic DNA from the parasites shown digested with EcoRV or BglII and probed with PrB. Sizes of fragments are in kbp.

Figure 2

Expression and binding of EBA-175 in P. falciparum lines to erythrocyte proteins. (A) Western blot of supernatants from W2mef, W2mefΔ175/1 and 2, 3D7, 3D7Δ175/1, and 3D7Δ175/1 and 2 probed with anti-EBA-175 (Upper) Abs (17). (Lower) A control of the same supernatants probed with anti-SERA5 Abs. (B) Western blot of supernatants from W2mef, W2mefΔ175/3′, 3D7, and 3D7Δ175/3′ probed with anti-EBA-175 Abs (Top) or 3′-Cys Abs (Middle). A control of the same supernatants is shown probed with anti-SERA5 (Bottom). (C) Membrane proteins from erythrocyte ghosts separated by SDS/PAGE and blotted to nitrocellulose. The filters were incubated with supernatants from 3D7, 3D7Δ175/3′, 3D7Δ175/1, W2mef, W2mefΔ175/3′, W2mefΔ175/1, or no supernatant (None). After washing, the incubated filter was probed with anti-EBA-175 Abs to detect bound ligand. The track on the right shows the erythrocyte membrane proteins probed with anti-glycophorin A/B Abs to show the glycophorin homodimer (A/A), glycophorin A and B heterodimer (A/B), and the glycophorin B homodimer (B/B). (D) Erythrocyte ghost proteins prepared as in C and incubated with the same supernatants but probed with anti-EBA-140 (BAEBL) Abs. The track labeled “None” was not incubated with supernatant but probed with anti-EBA-140 Abs to show nonspecific crossreactivity of the Ab with erythrocyte membrane proteins. The track on the right is the erythrocyte ghost proteins probed with anti-glycophorin C.

Figure 3

Comparison of merozoite invasion of mutant P. falciparum into enzyme-treated erythrocytes. Shown is merozoite invasion of W2mef, W2mefΔ175/1, W2mefΔ175/2, W2mefΔ175/3′, 3D7, 3D7Δ175/1, 3D7Δ175/2, and 3D7Δ175/3′ into neuraminidase- (A), trypsin- (B), or chymotrypsin (C)-treated erythrocytes. Invasion shown is relative to invasion of parental controls into untreated erythrocytes with 95% confidence levels indicated. Values represent 4–10 independent experiments, each done in triplicate. Assays were plated at 0.5% parasitemia, and the invasion rate for untreated erythrocytes was between 3% and 7% when sampled.

Figure 4

A model for invasion via sialic acid-dependent and -independent pathways in P. falciparum. The erythrocyte and invading merozoite are shown for W2mef, 3D7 W2mefΔ175, and 3D7Δ175. The red blocks at the apical end of each merozoite represent interaction with the PfRh proteins with their specific ligands, and this ligand–receptor interaction is a prerequisite for tight junction formation, perhaps by inducing release of EBA-175 and other ligands from the micronemes (22). After apical interaction the tight junction is formed by recruitment of high-affinity ligands such as EBA-175 that bind to the chymotrypsin-resistant receptor glycophorin A. In W2mef, EBA-175 is the dominant ligand, treatment of erythrocytes with neuraminidase (Neur-treated) removes all sialic acid, and the only ligands that can interact are sialic acid-independent. For W2mef there are not enough sialic acid-independent ligands to reach the required threshold of affinity to mediate invasion of neuraminidase-treated erythrocytes. Because glycophorin A is chymotrypsin-resistant, W2mef is able to invade erythrocytes treated with this protease through the EBA-175/glycophorin A pathway. 3D7 invades by using neuraminidase-independent pathways; however, EBA-175 is functional, so the parasite can invade both chymotrypsin- and neuraminidase-treated erythrocytes efficiently. The W2mefΔ175 and 3D7Δ175 parasites lacking EBA-175 show the same invasion phenotypes. They invade neuraminidase-treated erythrocytes through the ligand that replaced EBA-175 but cannot, however, invade chymotrypsin-treated erythrocytes because of the absence of EBA-175.