Plasmodium falciparum uses a key functional site in complement receptor type-1 for invasion of human erythrocytes

Abstract

The Plasmodium falciparum adhesin PfRh4 binds to complement receptor type-1 (CR1) on human erythrocytes and mediates a glycophorin-independent invasion pathway. CR1 is a complement regulator and immune-adherence receptor on erythrocytes required for shuttling of C3b/C4b-opsonized particles to liver and spleen for phagocytosis. Using recombinant CR1 constructs, we mapped the recognition site for PfRh4 to complement control protein modules 1 to 3 (CCP1-3) at the membrane-distal amino terminus of CR1. This region of CR1 binds to C4b and C3b and accelerates decay of both classic pathway and alternative pathway C3 and C5 convertases. CCP1-3 competed for PfRh4 binding to erythroid CR1 and inhibited the PfRh4-CR1 invasion pathways across a wide range of P falciparum strains. PfRh4 did not bind significantly to other CR1 constructs, including CCP15-17, which is 85% identical to CCP1-3. PfRh4 binding to CR1 did not affect its C3b/C4b binding capability, and we show evidence for a ternary complex between CCP1-3, C4b, and PfRh4. PfRh4 binding specifically inhibited CR1's convertase decay-accelerating activity, whereas there was no effect on factor H-mediated decay-accelerating activity. These results increase our understanding of the functional implications of CR1 engagement with PfRh4 and highlight the interplay between complement regulation and infection.

Figure 1

PfRh4 interacts with the N-terminal 3 CCPs of CR1. (A) Schematic of CR1 polypeptide. Each box labeled 1 to 30 represents a complement control protein module (CCP) of 60-70 amino acid residues. The first 28 CCPs are organized based on homology into 4 long homologous repeats (LHRs) A-D, each consisting of 7 CCPs. The functional sites of CR1 are labeled site 1 and site 2. The recombinant CR1 fragments used in this study are indicated as black bars with relevant CCPs labeled above. NH2 indicates amino terminus; TM, transmembrane domain; and CYT, cytoplasmic tail. (B) Recombinant PfRh4 binds to CCP1-3. An ELISA was used to measure the interaction between CR1 fragments and rPfRh4. Microtiter wells were coated with CCP1-3, CCP10-11, CCP15-17, CCP21-22, or CCP15-25 at 0.5 μg/well. Recombinant PfRh4 was added at 0.5 μg/well. Bound PfRh4 was detected with an anti-PfRh4 monoclonal antibody 10C9. (C) Recombinant PfRh4 forms a complex with CCP1-3 but not CCP15-17. Immunoprecipitation experiments were performed in which combinations of recombinant proteins CCP1-3/rPfRh4 or CCP15-17/rPfRh4 were incubated with an anti-CR1 monoclonal antibody 1B4. For Western blot analyses of immunoprecipitated eluates, soluble CR1 fragments were detected with anti-CR1 monoclonal antibody 1B4 and rPfRh4 was detected with 10C9 monoclonal antibody. (D) Binding of sCR1 to rPfRh4 was inhibited by CCP1-3. Microtiter plates were coated with saturating concentrations of rPfRh4 (5 μg/well). CCP1-3 or CCP15-17 at concentrations of 0nM, 0.02nM, 0.23nM, 2.3nM, 23nM, or 234nM was incubated with sCR1 (23nM) before addition to wells. Interaction between sCR1 and PfRh4 was detected using anti-CR1 antibody HB8592, which detected sCR1 and not smaller CR1 fragments. ELISA experiments in panels B and D were repeated with similar results and their y-axis represent A_{405 nm} with error bars showing the range of duplicate readings.

Figure 2

Delineation of PfRh4 binding sites on CR1 and K_D measurements by SPR. (A) Recombinant PfRh4 binds to sCR1 and CCP1-3 as shown by SPR. Duplicate injections of sCR1 and smaller CR1 fragments, all at a concentration of 5μM, were performed over a CM5 chip that was coupled with rPfRh4. A small fraction of inactive rPfRh4 present on the chip surface triggers nonspecific background binding for the first analyte samples to be assayed (CCP1-3 and sCR1), as manifested in imperfect reproducibility of the sensorgrams. After saturation of this nonspecific binding capacity, duplicate injections are of acceptable reproducibility (all other constructs). (B) Use of SPR to measure the dissociation constant (K_D) of CCP1-3 for PfRh4. The left-hand panels show duplicate sensorgrams for a range of increasing CCP1-3 concentrations (1.0μM, 2.5μM, 5.0μM, 10.0μM, and 25.0μM, bottom to top) flowing over a CM5-chip surface with a loading of 410 RUs (i) and 1480 RUs (iii) of rPfRh4. The right-hand panels show plots of the RUs versus CCP1-3 on 2 different flow cells, coupled with 410 RUs (ii) and 1480 RUs (iv) of rPfRh4. The dashed vertical line indicates the K_D fitted to both plots simultaneously. In all panels, blank-subtracted sensorgrams are shown.

Figure 3

CCP1-3 inhibits native PfRh4 erythrocyte binding and the CR1-PfRh4 invasion pathway. (A) Native PfRh4 binding to erythroid CR1 was inhibited by CCP1-3. Competitive binding assays were performed by incubating either CCP1-3 or CCP15-17 with invasion supernatants at the stated final concentrations of 4 μg/mL or 8 μg/mL. The black numbers in the top panel represent the percentage of PfRh4 binding relative to PBS for each concentration as determined by densitometry. Immunodetection of parasite proteins with anti-PfRh4 or anti-EBA-175 antibodies after erythrocyte binding is shown. (B) The PfRh4 invasion pathway was inhibited in the presence of CCP1-3. Parasite strains W2mefΔRh4 (gray bars, untreated erythrocytes) and 3D7 (black bars for untreated, white bars for nm-treated erythrocytes) were tested in growth assays in the presence of final concentrations of 0.5 mg/mL CCP1-3, CCP10-11, CCP15-17, CCP21-22, CCP15-25, or sCR1. Growth (percentage of control) on the y-axis refers to the percentage of parasitemia in the presence of CR1 constructs relative to the percentage of parasitemia with the addition of PBS (arbitrarily set to be 100%).

Figure 4

PfRh4-CR1 pathway constitutes the majority of sialic acid-independent invasion events. (A) Sialic acid–dependent and –independent parasite strains. Parasite strains were assayed for their ability to invade nm-treated erythrocytes. The y-axis represents percentage of growth of parasites into nm-treated erythrocytes relative to growth of parasites into untreated erythrocytes. (B) Expression of PfRh4 in P falciparum strains. Western blot of saponin-lysed schizont pellets probed with anti-PfRh4 monoclonal antibody 2E8 (top panel) and anti–EBA-175 rabbit polyclonal antibody (bottom panel). (C) Growth of sialic acid–independent strains in untreated erythrocytes was slightly inhibited in the presence of CCP1-3. Parasite strains were tested in growth assays into untreated erythrocytes in the presence of 0.5 mg/mL final concentration of CCP1-3 (white bars) or BSA (black bars). Growth (percentage of control) on the y-axis refers to the percentage parasitemia in the presence of CCP1-3 or BSA relative to the percentage parasitemia with the addition of PBS, which is arbitrarily set at 100%. (D) Growth of sialic acid-independent strains in nm-treated erythrocytes was inhibited in the presence of CCP1-3. Parasite strains were tested in growth assays using nm-treated erythrocytes in the presence of 0.5 mg/mL CCP1-3 (white bars) or BSA (black bars).

Figure 5

C3b/C4b interaction with CR1 is not perturbed by PfRh4 binding. (A) PfRh4 binding does not affect C3b interaction with sCR1. Duplicate injections of a concentration series of sCR1 (i) or a mixture of sCR1 plus 3-fold molar excess of rPfRh4 (iii) onto a CM5 chip coupled with C3b. K_D measurements of sCR1:C3b complex alone (ii) or in the presence of 3-fold molar excess of rPfRh4 (iv) with fitted value indicated by dotted line (B) PfRh4 binding does not affect C4b interaction with sCR1. Duplicate injections of a concentration series of sCR1 (i) or a mixture of sCR1 plus 3-fold molar excess of rPfRh4 (iii) onto a CM5 chip coupled with C4b. K_D measurements of sCR1:C4b complex alone (ii) or in the presence of 3-fold molar excess of rPfRh4 (iv) with fitted value indicated by dotted line. In all panels, blank-subtracted sensorgrams are shown. (C) Native PfRh4 binding to erythroid CR1 was not inhibited by C3b or C4b. Competitive binding assays were performed by incubating either C3b or C4b with invasion supernatants at the stated final concentrations (8 μg/mL or 16 μg/mL). Immunodetection of parasite proteins with anti-PfRh4 and anti–EBA-175 antibodies after erythrocyte binding is shown. (D) PfRh4 invasion pathway was not inhibited in the presence of C3b or C4b. Parasite strains W2mefΔRh4 (gray bars) and 3D7 (black bars for untreated, white bars for nm-treated) were tested in growth assays in the presence of 0.5 mg/mL C3b or C4b. Growth (percentage of control) on the y-axis refers to the percentage parasitemia in the presence of CR1 constructs relative to the percent parasitemia with the addition of PBS (arbitrarily set to be 100%).

Figure 6

A ternary complex composed of CCP1-3, C4b, and PfRh4. (A) ELISA of ternary complex. Microtiter plates were coated with C4b (1 μg/well). After incubation with rPfRh4, CCP1-3, or both as indicated, bound rPfRh4 was detected using anti-PfRh4 monoclonal antibody 10C9 (black bars) that was raised against rPfRh4. Monoclonal antibody 2E8 (white bars) was raised to the C-terminal end of PfRh4 and recognizes native PfRh4 but not the region encompassed by rPfRh4. The inset shows the reactivity of these monoclonal antibodies in an ELISA using microtiter plates coated with rPfRh4. Data are the mean ± SD for 3 independent experiments. (B) Immunoprecipitation of ternary complex. Purified C4b, CCP1-3, or rPfRh4 were incubated together as indicated (at 0.02 mg/mL). Western blots were performed after immunoprecipitation with anti-PfRh4 10C9 (lanes 1-3) or 2E8 (lane 4) monoclonal antibody, respectively. Immunoprecipitated material was probed with a rabbit polyclonal anti-PfRh4 antibody, a goat polyclonal anti–human C4 antibody and a monoclonal anti-CR1 antibody 1B4, respectively. PfRh4 and C4b samples were run under reducing conditions whereas CCP1-3 was under nonreducing conditions. Arrowheads highlight specific protein bands. Western blot results are representative of 2 or 3 independent experiments. (C) A ternary complex composed of CCP1-3, C4b, and rPfRh4 as inferred from SPR. Measurement by SPR of CCP1-3 binding to C4b immobilized on a CM5 sensorchip in absence (blue lines) and presence (black lines) of a 2-fold molar excess of rPfRh4. CCP1-3 concentrations were 4μM, 2μM, 1μM, 0.1μM, and 0.05μM; single measurements were made at 4μM and 1μM while all others were duplicate measurements. (D) Plots of responses versus concentration of CCP1-3 (either alone, blue, or mixed with rPfRh4, red) from the sensorgrams in panel C are shown, with extrapolations to R_max values. The much higher responses obtained when rPfRh4 is coinjected with CCP1-3 are consistent with formation of ternary complexes rather than binary complexes. (E) The percentage of maximal binding (estimated from extrapolated R_max values in panel D) versus concentration of CCP1-3 (either alone, blue, or mixed with rPfRh4, red). The near identical-slope of both binding curves indicates that the affinity of CCP1-3 for C4b, while it cannot be quantified because of subsaturation concentrations of CCP1-3, is not radically altered by rPfRh4 and is consistent with a ternary complex in which C4b and rPfRh4 occupy distinct sites on CCP1-3.

Figure 7

PfRh4 disrupts CR1's decay accelerating activity. (A) Decay acceleration of C3bBb was inhibited on PfRh4 binding. SPR was used to monitor formation of the C3bBb convertase complex as factor D and factor B were flowed together over C3b that was amine-coupled to a CM5 sensor chip (i, C3bBb formation). The subsequent decline in response reflects decay of the complex as Bb is released from the chip surface (ii, spontaneous decay). The rate of decay was accelerated by initiating a flow of CCP1-3 or factor H CCP1-4 (FH1-4; iii, analyte injection), but not when CCP1-3 was in the presence of 3-fold molar excess of rPfRh4. In panels A-D, any convertases remaining were decayed by injecting FH1-4 at ∼ 800 seconds to aid regeneration (B) Decay-acceleration of CCP1-3 was affected in a dose-dependent manner by rPfRh4. The rate of decay was monitored in the presence of increasing concentrations of rPfRh4. A small fraction of inactive rPfRh4 present on the surface triggers nonspecific background binding when the first duplicate injection was assayed. (CCP1-3 in 3-fold molar excess of PfRh4) as manifested in the imperfect reproducibility of the sensorgrams. After saturation of this nonspecific binding capacity, injections were of acceptable reproducibility (all other constructs). (C) Decay-acceleration activity of sCR1 was affected by presence of rPfRh4. Shown are binding responses for sCR1, and for sCR1 in the presence of a 5-fold molar excess of PfRh4, of the convertase C3bBb (+C) or C3b alone (−C). Three C3b-binding sites in sCR1 mediate the overall high binding levels to both the convertase and C3b alone. Only sCR1 binding to the convertase (+C) shows a distinctive association curve that is consistent with an initially enhanced binding of sCR1 to the convertase, followed by 2 simultaneous, overlapping processes: accelerated decay of C3bBb into Bb and surface-bound C3b, and binding of sCR1 to C3b. (D) Decay acceleration by factor H CCP1-4 was not affected by rPfRh4. Assays shown in panels A and C, and in panels B and D were performed on identical biosensor surfaces. (E) Hemolysis assay for the classic pathway. The classic pathway C3 convertase was assembled on the surface of antibody-sensitized sheep erythrocytes. PfRh4 plus CCP1-3 were pre-incubated before mixing with sheep erythrocytes. Cellular lysis was induced by the addition of guinea pig serum and monitored by the O.D. of the supernatant at 414nM. CCP1-3 was used at a concentration of 6.8nM. (F) Hemolysis assay for the alternative pathway. The alternative pathway convertase was prepared using EAC14 cells by the addition of C2 and C3. PfRh4 plus CCP1-3 were preincubated and these mixtures were then added to EAC43 cells and the alternative pathway components factor B, factor D, and properdin and were added and lysis measured as in panel E. A convertase was not formed in the absence of factor B. CCP1-3 was used at a concentration of 34nM. For all panels, **P < .01; ***P < .001).