Shed EBA-175 mediates red blood cell clustering that enhances malaria parasite growth and enables immune evasion
- Washington University School of Medicine, United States
- National Institute of Allergy and Infectious Diseases, National Institutes of Health, United States
- University of Copenhagen, Denmark
Figures
Figure 1 with 1 supplement
Native EBA175 promotes RBC clustering.
(A-C) represent data from one biological replicate. (A) One representative Coomassie brilliant blue stained gel of immunopreciptations. Lane 1, control Ab Ab-8C2, lane 2, anti-EBA175 Ab-218. (B) Pseudocolor dot plots of the IP-elutions showing RBC clustering as observed by increasing forward scatter (FSC-A) and side scatter (SSC-A). FSC-A is correlated with the size or volume of an object while SSC-A is a measure of the internal composition of the object. Frequency of events (clusters) out of one million counts are located in bottom right corner of dot plot for one representative technical replicate. (C) Frequency of events (clusters) seen in the FSC-A gated population for the IP-elutions, ●=Control, ■=EBA-175 treated. Values shown are for five technical replicates of one biological replicate. (Two additional biological replicates each consisting of five technical replicates can be found in Figure 1—figure supplement 1) (D) RBC clusters are observed in parasite culture as observed by FSC-A. Top panel - culture viability as shown by GFP-A for one technical replicate. Bottom panel - overall size difference of each culture as shown by FSC-A and the gated populations for one technical replicate. Frequency of events (clusters) out of one million counts are located in bottom right corner of dot plot for one representative technical replicate. (E) Median GFP-A signal of the total population (top) of five biological replicates each consisting of three technical replicates and frequency of events (clusters) in the gated population for five biological replicates (bottom) showing GpA dependent clustering.
Figure 1—figure supplement 1
Native EBA-175.
(A) Domain structure of EBA-175. SP-signal peptide, RII-region II composed of F1 and F2 domains, VI-region VI, TM-transmembrane domain, salmon colored domains-cytoplasmic domains. (B) Western blot of the control elution (left lane) and αEBA-175 elution (right lane) using an EBA-175 specific antibody. (C) Unique peptides identified by mass spectrometry for both the control and αEBA-175 immunoprecipitations. (D) Peptide coverage of the αEBA-175 immuno-precipitations results mapped onto the sequence of EBA-175 (PF3D7_0731500), Bold – RII sequence, Red – amino acids identified by mass spectrometry, Blue – site of cleavage by PfROM4 (16) (E) Coomassie stained protein gels and gated counts for two additional purifications, with five technical replicates, for each immunoprecipitation elution incubated with RBCs.
Figure 2 with 2 supplements
EBA-175 RII binds and promotes RBCs to form clusters through interactions with GpA.
(A) Clustered RBCs adhere to one another via RII at 300pM and 1 nM concentrations. Scale bars are 10 µm. (B) RII, but not the individual DBL domains F1 or F2, induces RBC clustering in a concentration dependent manner as observed by FSC-A. Frequency of events (clusters) out of 100,000 counts are located in bottom right corner of dot plot for each sample when gated according to the no protein control (top left). One representative biological replicate out of three is presented. (C) Clustered RBCs adhere to one another via RII at the interface between each red cell. Scale bars are 10 µm. (D) Inhibition of particle size shift of RBCs incubated with RII by neuraminidase (NA, panel 2), anti-GpA Ab (panel 3 and 4), and anti-RII Ab-217 as observed by FSC-A (panel 5 and 6). Frequency of events (clusters) out of 100,000 counts are located in bottom right corner of dot plot for each sample when gated according to the no protein control (top left). One representative biological replicate out of three is presented. (E) Analysis of cluster size for varying concentrations of EBA-175 RII under flow conditions of 1 dyn/cm2. Left hand panel shows representative data from one experiment. Each circle represents a single cluster area (µm2). Middle panel shows mean cluster size for each concentration, and right hand panel shows mean number of clusters per concentration for three independent experiments ± S.D.
Figure 2—figure supplement 1
EBA-175-RII mediates similar levels of parasite growth in O, A, and B blood types.
(A) RII induced RBC clusters in O, A, and B blood as observed by increasing FSC-A and SSC-A in the presence or absence of Ab-217. (B) The number of clusters was quantified by gating according to the control with no protein added, values are for nine biological replicates of one million counts each for each condition. Results are shown as ±SEM. ○=untreated RBCs, □=30 nM RII, Δ = 30 nM RII/300 nM Ab-217, black = O blood, white = A blood, dark gray = B blood. (C) RII-induced growth enhancement of NF54-GFP-luc parasites in O, A, and B blood types, in the presence Ab-217 or Ab-218, as assessed by luciferase assay. Data shown are mean ± SD of three technical replicates.
Figure 2—figure supplement 2
RII binds RBCs and forms stable clusters under flow conditions.
(A) Inhibition of RII induced size shift of RBCs incubated with RII by neuraminidase, anti-glycophorin A Ab, and anti-RII Ab-217 and treatment controls without RII-175. FSC-A is correlated with the size or volume of an object while SSC-A is a measure of the internal composition of the object. Frequency of events (clusters) out of 100,000 counts are located in bottom right corner of dot plot for each sample when gated according to the no protein control (B) Representative images of RBCs under physiological flow conditions at various concentrations of RII. Scale bars are 10 µm.
Figure 3 with 2 supplements
Recombinant EBA-175 RII causes RBC clustering in parasite culture and enhances parasite growth.
(A) Effects of picomolar to low nanomolar RII on parasite growth assessed by [3H]-hypoxanthine uptake for 3D7, Dd2, FVO/FCR1, HB3 strains, and the luciferase activity for NF54-GFP-luc strain (far right). Data shown are mean ± SD of three biological replicates for all strains. Significance (*, p<0.05; ***, p<0.001) (B) Uptake of [3H]-hypoxanthine into parasite nucleic acids by 3D7, Dd2, FVO/FCR1, HB3 strains, and the luciferase activity in NF54-GFP-luc strain (far right), in the presence or absence of RII, F1 or F2 at varying concentrations (10 nM – 1 μM range) for 96 hr. Results are shown as mean ± SD of three biological replicates. (C) Ab-217 (purple line) inhibition of RII-mediated growth enhancement, control Ab Ab-DBP (black line). Results are shown as mean ± SD of three biological replicates. (D) Microscopy analysis of NF54-GFP parasites (green) in RII-induced clusters (red).
Figure 3—figure supplement 1
RII enhances parasite growth.
(A) The distribution of the three biological replicates of growth curves for P. falciparum strains 3D7, Dd2, FVO/FCR1, HB3, and NF54-GFP-luc incubated with increasing concentrations of F1, F2, or RII. In accordance with ALARA for the use and disposal of radioactive waste, two technical replicates in each biological replicate were performed for [3H]-hypoxanthine incorporation assays. Three technical replicates for each biological replicate were assessed in the luciferase activity analysis. (B) The proliferation/growth of parasites was assessed by measuring [3H]-hypoxanthine uptake or luciferase activity (NF54-GFP-luc strain) in the presence or absence of 30 nM F1, F2 or RII. Data shown are mean ± SD of ten replicates and statistical analysis was performed as described in Materials and methods.
Figure 3—figure supplement 2
Parasite growth enhancement is due to the specific interaction of EBA-175 with GpA.
(A) The distribution of the three biological replicates of various P. falciparum cultures left untreated (red circle) or treated with 30 nM RII for 96 hr in the presence of various concentrations Ab-DBP (black line) or Ab-217 (purple). Growth of 3D7, Dd2, FVO/FCR1 and HB3 cultures were assessed by measuring [3H]-hypoxanthine uptake and NF54-GFP-luc culture by measuring luciferase activity. In accordance with ALARA for the use and disposal of radioactive waste, two technical replicates in each biological replicate were performed for [3H]-hypoxanthine incorporation assays. Three technical replicates for each biological replicate were assessed in the luciferase activity analysis. (B) P. falciparum 3D7, Dd2, FVO/FCR1, HB3, and NF54-GFP-luc cultures were left untreated (control) or treated with 30 nM RII for 96 hr in the presence of 1 μM Ab-DBP or Ab-217. The proliferation/growth of parasites was assessed by measuring [3H]-hypoxanthine uptake or, in the case of NF54-GFP-Luc, luciferase activity. Ab-217 significantly blocked the RII-mediated growth enhancement while Ab-DBP had no effect. Results are shown as mean ± SD of six technical replicates and statistical analysis was performed as described in Materials and methods.
Figure 4 with 1 supplement
EBA-175 RII-mediated RBC clustering provides parasites a growth advantage and confers protection from anti-AMA1 and anti-RH5 neutralizing antibodies.
(A–C) The presence of 30 nM RII in (A) 3D7, (B) FVO/FCR1, and (C) NF54-GFP-luc cultures mitigated the inhibitory effects of the anti-AMA1 neutralizing antibodies N3-2D9 and N4-1F6 at 10 mg/mL. (D–F) A 30 nM RII mitigated the ability of anti-RH5 neutralizing Ab, 9AD4, to inhibit growth of (D) 3D7, (E) FVO/FCR1 and (F) Dd2 cultures at various concentrations. Results are shown as mean ± SD of three biological replicates and significance was determined as described in Materials and methods.
Figure 4—figure supplement 1
EBA-175-mediated RBC clustering confers parasites a survival advantage by preventing neutralization by antibody 9AD4.
(A) The distribution of the three biological replicates illustrating that the presence of 30 nM RII mitigated the inhibitory effects of anti-RH5 neutralizing antibody, 9AD4, on 3D7, FVO/FCR1 and Dd2 culture growth at various antibody concentrations. In accordance with ALARA for the use and disposal of radioactive waste, two technical replicates in each biological replicate were performed for [3H]-hypoxanthine incorporation assays whereas. Technical replicates for each biological replicate were assessed in the luciferase activity analysis. (B) 3D7, FVO/FCR1 and Dd2 cultures were untreated (control – open circles) or treated with various concentrations of anti-RH5 antibody 9AD4 in the absence (closed circles) or presence (red squares) of 30 nM RII for 96 hr. Left panel – 0.01 mg/mL 9AD4, Middle panel – 0.1 mg/mL 9AD4, Right panel – 1.0 mg/mL 9AD4. The parasite growth was assessed by measuring [3H]-hypoxanthine incorporation. Results are shown as mean ± SD of eight technical replicates and statistical analysis was performed as described in Materials and methods.
Figure 5
Model of the parasite survival and immune evasion mediated by EBA-175.
(A) Merozoite (purple) secretes EBA-175 to the apical end for engagement of GpA. (B) Post invasion, EBA-175 is cleaved from the surface and shed. (C) Shed EBA-175 diffuses to neighboring RBCs and engages GpA resulting in RBC recruitment and red cell clustering (D). (E) RBC clustering enables daughter merozoites easy access to an uninfected RBC for the next round of invasion. Gray arrow indicates progression through one round of invasion and replication.
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Shed EBA-175 mediates red blood cell clustering that enhances malaria parasite growth and enables immune evasion
eLife 7:e43224.
https://doi.org/10.7554/eLife.43224
