Reticulocyte and erythrocyte binding-like proteins function cooperatively in invasion of human erythrocytes by malaria parasites

Abstract

Plasmodium falciparum causes the most severe form of malaria in humans and invades erythrocytes using multiple ligand-receptor interactions. Two important protein families involved in erythrocyte binding are the erythrocyte binding-like (EBL) and the reticulocyte binding-like (RBL or P. falciparum Rh [PfRh]) proteins. We constructed P. falciparum lines lacking expression of EBL proteins by creating single and double knockouts of the corresponding genes for eba-175, eba-181, and eba-140 and show that the EBL and PfRh proteins function cooperatively, consistent with them playing a similar role in merozoite invasion. We provide evidence that PfRh and EBL proteins functionally interact, as loss of function of EBA-181 ablates the ability of PfRh2a/b protein antibodies to inhibit merozoite invasion. Additionally, loss of function of some ebl genes results in selection for increased transcription of the PfRh family. This provides a rational basis for considering PfRh and EBL proteins for use as a combination vaccine against P. falciparum. We immunized rabbits with combinations of PfRh and EBL proteins to test the ability of antibodies to block merozoite invasion in growth inhibition assays. A combination of EBA-175, PfRh2a/b, and PfRh4 recombinant proteins induced antibodies that potently blocked merozoite invasion. This validates the use of a combination of these ligands as a potential vaccine that would have broad activity against P. falciparum.

FIG. 1.

Genetic disruption of the eba-175, eba-181, and eba-140 genes in P. falciparum. (A) The strategy for construction of 3D7 P. falciparum parasites with single- and double-gene knockouts of the ebl family. The vectors pCC1 and pCC4 were used, allowing selection for hDHFR (WR99210) and blasticidin deaminase (blasticidin), respectively. Flucytosine (5FC) was used for negative selection to obtain parasites with a double-crossover recombination event with Saccharomyces cerevisiae cytosine deaminase/uracil phosphoribosyltransferase. The eba-175 and eba-181 genes were disrupted first using pCC1 and then using eba-140 or eba-181 with pCC4. (B) The eba-140 gene was disrupted in 3D7 using pCC4 to make a single knockout by double-crossover recombination. (C) Southern blot analysis of 3D7, 3D7Δ175, 3D7Δ181, 3D7Δ140, 3D7Δ175/181, 3D7Δ175/140, and 3D7Δ181/140 to show that pCC1 and pCC4 had integrated into the expected genes by double-crossover recombination. For the first panel, the restriction enzymes used were EcoRV and MfeI (EBA-175), while for the second panel they were EcoRI and NcoI (eba-181) and for the third panel they were AgeI, HincII, and BamHI (EBA-140). (D) Immunoblots using specific antibodies to EBA-175, EBA-181, and EBA-140 to show that the different knockout strains lacked expression of the expected proteins. Shown are supernatants harvested from 3D7, 3D7Δ175, 3D7Δ181, 3D7Δ140, 3D7Δ175/181, 3D7Δ175/140, and 3D7Δ181/140 that were probed with anti-EBA175, anti-EBA-181, or anti-EBA-140 antibodies.

FIG. 2.

Structures of the EBA-175, EBA-181, EBA-140, PfRh2a, PfRh2b, and PfRh4 proteins and the positions to which recombinant proteins were made for use as immunogens to raise antibodies. The red region in EBA-175, EBA-181, and EBA-140 corresponds to region II, which encompasses the receptor-binding domain. PfRh2a and PfRh2b are identical for most of the N-terminal region and differ only at the C terminus (green for PfRh2a and blue for PfRh2b). The red cell binding region defined for PfRh4 is shown in yellow, toward the N terminus. The proteins are drawn to scale, with the sizes shown in amino acids (aa).

FIG. 3.

Antibodies to EBL and PfRh2a/b proteins inhibit invasion of P. falciparum into human erythrocytes. The results of GIAs for 3D7, 3D7Δ175, 3D7Δ175/181, 3D7Δ181, 3D7Δ175/140, 3D7Δ140, 3D7Δ175/140, and 3D7Δ175/181 are shown. The antibodies used are IgG purified from rabbits, used at 1 mg/ml. Shown are the results of three independent experiments, with each done in triplicate, using anti-EBA-175, anti-EBA-181, anti-EBA-140, and anti-PfRh2a/b antibodies which were made to the regions of each protein, as shown in Fig. 2. The histogram represents 3 independent experiments, performed in triplicate. The error bars show the standard errors of the means.

FIG. 4.

Antibody titers for the IgG antibodies purified from rabbits immunized with the EBA-175, PfRh2a/b, and PfRh4 combinations. (A) PfRh2a/b and EBA-175 recombinant proteins were coated on separate plates, and antibodies from rabbits were tested for reactivity. Prebleed antibodies and PBS were used as negative controls. (B) PfRh4 and EBA-175 recombinant proteins were coated on separate plates, and antibodies from rabbits were tested for reactivity. Prebleed antibodies and PBS were used as negative controls. (C) PfRh4, PfRh2a/b, and EBA-175 recombinant proteins were coated on separate plates, and antibodies from rabbits were tested for reactivity. Prebleed antibodies and PBS were used as negative controls.

FIG. 5.

Antibodies react with the corresponding EBL and PfRh proteins in immunoblots of supernatants or schizont preparations made from 3D7, 3D7Δ175, FCR3, W2mefΔRh4, W2mef, and W2mefΔ175. (A) Supernatants from 3D7, 3D7Δ175, FCR3, and W2mefΔRh4 were separated by SDS-PAGE, and filters were probed with antibodies from rabbits immunized with EBA-175. 3D7Δ175 lacks expression of EBA-175. (B) Supernatants from 3D7, 3D7Δ175, FCR3, and W2mefΔRh4 were separated by SDS-PAGE, and filters were probed with antibodies from rabbits immunized with EBA-175 and PfRh2a/b. (C) Supernatants from 3D7, 3D7Δ175, FCR3, and W2mefΔRh4 were separated by SDS-PAGE, and filters were probed with antibodies from rabbits immunized with EBA-175, PfRh2a/b, and PfRh4. FCR3 lacks expression of PfRh2a and PfRh2b, while 3D7Δ175 does not express EBA-175. W2mefΔRh4 does not express PfRh4. (D) Schizonts from W2mef, W2mefΔ175, W2mefΔRh4, and 3D7 were separated by SDS-PAGE, and filters were probed with antibodies from rabbits immunized with EBA-175, PfRh2a/b, and PfRh4. W2mefΔ175 does not express EBA-175 but does express PfRh4. W2mefΔRh4 does not express PfRh4 but does express EBA-175. W2mef does not express PfRh4.

FIG. 6.

Antibodies from rabbits immunized with EBA-175, PfRh2a/b, and PfRh4 inhibit growth of P. falciparum. (A) Results of GIAs of 3D7 and 3D7Δ175 using IgG antibodies from rabbits immunized with a combination of EBA-175 and PfRh2a/b. (B) Titration of antibodies from rabbits immunized with a combination of EBA-175 and PfRh2a/b tested in GIAs with 3D7 and 3D7Δ175 parasites. (C) Results of GIAs of 3D7 and 3D7Δ175 using IgG antibodies from rabbits immunized with a combination of EBA-175 and PfRh4. (D) Titration of antibodies from rabbits immunized with a combination of EBA-175 and PfRh4 tested in GIAs with 3D7 and 3D7Δ175 parasites. (E) Results of GIAs of 3D7 and 3D7Δ175 using IgG antibodies from rabbits immunized with a combination of EBA-175/PfRh4 and PfRh2a/b. (F) Titration of antibodies from rabbits immunized with a combination of EBA-175, PfRh4, and PfRh2a/b tested in GIAs with 3D7 and 3D7Δ175 parasites.

FIG. 7.

P. falciparum lines lacking expression of specific EBL proteins are less fit than the parental line 3D7, as shown in growth competition assays. The parasite lines 3D7Δ175, 3D7Δ181, 3D7Δ140, 3D7Δ175/181, and 3D7Δ175/140 were mixed in approximately equal amounts with the 3D7 parental line, and the growth of each was monitored by qPCR over a 5-week period. The log₁₀ ratio of the parent/knockout (KO) strain was calculated. The P values shown represent the probability of a difference in growth rate compared to the 3D7 growth rate and were calculated using a Student t test.

FIG. 8.

3D7 ebl gene-knockout lines vary in the relative expression of PfRh4 in late-stage schizonts. The genes whose transcriptional levels were compared included actin, eba-140, eba-175, eba-181, mtrap, PfRh1, PfRh2a, PfRh2b, PfRh4, ron5, and PfRh5. The histograms show the relative concentration (knockout strain/3D7) as a ratio for the 11 genes used. Values are given as means ± standard errors of the means. The statistical analysis used was the Student t test. The bars show the results of three independent experiments done in triplicate. (A) Comparison of transcriptional levels between 3D7 and 3D7Δ175 (which lacks expression of EBA-175); (B) comparison of transcriptional levels between 3D7 and 3D7Δ181 (which lacks expression of EBA-181); (C) comparison of transcriptional levels between 3D7 and 3D7Δ140 (which lacks expression of EBA-140); (D) comparison of transcriptional levels between 3D7 and 3D7Δ175/181 (which lacks expression of EBA-175 and EBA-181); (E) comparison of transcriptional levels between 3D7 and 3D7Δ175/140 (which lacks expression of EBA-175 and EBA-140).