Plasmodium falciparum causes the deadliest form of malaria, resulting in over a million deaths every year. Now, a team led by Alan F. Cowman has identified a gene that is used by this parasite to switch between the pathways it can use to invade red blood cells, and published their discovery in Science.

P. falciparum uses different pathways to invade the host erythrocyte. Some strains mainly use ligands that bind to erythrocyte receptors that contain sialic acid, whereas other strains use ligands that bind to erythrocyte receptors without sialic acid. Parasites of the W2mef P. falciparum strain can switch from sialic-acid-dependent to sialic-acid independent invasion. This can be achieved in the laboratory by selection on erythrocytes treated with neuraminidase, an enzyme that removes the sialic-acid residues, as well as by disruption of the sialic-acid-dependent ligand erythrocyte-binding antigen EBA175 (W2mefΔ175).

When selecting two clonal lines of W2mef (W2m/c2/N and W2m/c4/N) for sialic-acid-independent invasion on neuraminidase-treated erythrocytes, parasites were obtained that showed invasion of erythrocytes comparable to that of W2mefΔ175. When these parasites were grown on normal erythrocytes for several months, the clonal lines reverted to sialic-acid-dependent invasion. This ability to switch invasion pathways was reproducible.

To detect any shift in gene expression between W2mef, W2mefΔ175 and W2m/c4/N, Stubbs et al. used oligonucleotide arrays, and found that PfRh4 (P. falciparum reticulocyte-binding-like homologue 4) showed reproducible transcriptional differences. Using RT-PCR, they also found that transcription of PfRh4 increased about 60- to 80-fold in the sialic-acid-independent lines compared with the sialic-acid-dependent lines, suggesting that the activation of PfRh4 is required for switching from sialic-acid-independent invasion to sialic-acid-independent invasion. Accordingly, PfRh4-specific antibodies did not detect PfRh4 in sialic-acid-dependent parasites, whereas it was expressed in sialic-acid-independent parasites.

To confirm the role of PfRh4 in the switch from sialic-acid-independent to sialic-acid-independent invasion, transgenic parasites in which the PfRh4 gene was disrupted were grown on normal or neuraminidase-treated cells. These transgenic parasites were unable to switch to sialic-acid-independent invasion, so expression of the PfRh4 protein is required for this process. Finally, Cowman and colleagues determined the location of PfRh4 in the parasite by constructing transgenic parasite lines that expressed PfRh4 as a chimaeric protein with green fluorescent protein. They found that the protein is located at the apical tip of merozoites, which is consistent with a direct function of PfRh4 in invasion of erythrocytes.

This discovery of the role of PfRh4 in the switching of invasion strategies has important implications for anti-malaria vaccine design. A functional binding domain of the sialic-acid-dependent ligand EBA175 is currently being developed as a possible malaria vaccine. However, current efforts to target ligands that have a role in sialic-acid-dependent invasion might not be sufficient if the parasite can switch to a sialic-acid independent invasion pathway.