Human complement is the first line of defense against invading pathogens, including the malaria parasite Plasmodium falciparum. We previously demonstrated that human complement represents a particular threat for the clinically relevant blood stages of the parasite. To evade complement-mediated destruction, the parasites acquire factor H (FH) via specific receptors. We now report that the FH-related protein FHR-1 competes with FH for binding to the parasites. FHR-1, which is composed of five complement control protein domains with variable homology to FH but lacks C3b regulatory activity, accumulates on the surfaces of intraerythrocytic schizonts and free merozoites. Although binding of FH to schizont-infected RBCs and merozoites is increased in FHR-1–deficient human serum, the addition of recombinant FHR-1 decreases FH binding. The presence of FHR-1 consequently impairs C3b inactivation and parasite viability. We conclude that FHR-1 acts as a protective factor in human immunity by counteracting FH-mediated microbial complement evasion.

With more than 200 million cases annually, the mosquito-borne tropical disease malaria is a leading cause of death worldwide. Every year, an estimated 445,000 people die of malaria, and at particular risk are African children under five years of age. Global efforts to roll back malaria are undermined by the spread of parasite resistance against commonly used drugs, and, to date, no preventive vaccine is available (1, 2).

Malaria is caused by unicellular parasites of the genus Plasmodium, with P. falciparum being the predominant species in Africa. The blood stages of the infection are responsible for the clinical symptoms of malaria, which include fever, headache, anemia, and capillary sequestration. During the 48-hour replication cycle, merozoites (MZs) infect RBCs and develop into schizonts (SZs) containing 16–32 daughter-MZs, which are released into the circulation to infect new RBCs.

The intraerythrocytic lifestyle of Plasmodium parasites is a means to evade recognition by the human immune system. Human complement is one of the first immune components that the blood stages encounter, and, in particular, the alternative pathway of complement has a severe effect on parasite viability (37). To protect from complement-mediated lysis, both the extracellular MZs as well as the SZ-infected RBCs (SZ-iRBCs) acquire complement regulators, in particular factor H (FH) (68). FH is the major regulator of the alternative pathway and is able to inhibit the complement cascade by two activities, a cofactor activity during factor I–mediated C3b inactivation and a C3 convertase–specific decay-accelerating activity. FH consists of 20 short complement control protein domains (CCPs). Whereas CCP1–4 are involved in the regulatory activity of FH, CCP6–7 and CCP19–20 mediate binding to C3b and glycosaminoglycans on cellular surfaces (9) (Fig. 1A).

Our group previously observed that during P. falciparum infection, the SZ-iRBCs bind the FH-related (FHR) protein FHR-1 in addition to FH (7). FHR-1 is a member of the FHR family, comprising FHR-1 to FHR-5 (Fig. 1A) (10, 11). The genes encoding FH and the five FHR proteins are arranged in gene clusters, with the gene encoding FHR-1 being located directly next to the FHR-3–encoding gene. The cluster is prone to deletion events, and ∼15% of the world’s population carry a CFHR-3/CFHR-1 gene deletion in at least one allele (12). This mutation is associated with a number of autoimmune diseases, including atypical hemolytic uremic syndrome and age-related macular degeneration (13, 14).

Because none of the FHR proteins possess domains similar to the regulatory domains of FH, it was previously suggested that the proteins compete with FH for binding to cell surfaces but without regulating C3b, thereby fine-tuning the inhibitory role of FH in complement activation (11). In this study, we aimed to unveil the potential modulatory role of FHR-1 during complement evasion by the P. falciparum blood stages.

Expression of recombinant FHR-1 (rFHR-1) in Pichia pastoris was described before (15, 16). Generation of the polyclonal mouse antisera Pf39 (polyclonal Ab [pAb] Pf39) and rabbit anti–FHR-1 antisera (pAb FHR-1) was described elsewhere (17, 18). Monoclonal mouse anti-human FH Ab (mAb FH; isotype IgG2bκ, clone 131X, directed against CCP8-15), monoclonal mouse anti-SC5b9 Ab (mAb terminal complement complex [TCC]), monoclonal mouse anti-human inactivated C3b (iC3b) (mAb iC3b), and polyclonal goat anti-human C3 Ab (pAb C3) were purchased from Quidel.

Asexual blood stages of the P. falciparum gametocyte-deficient strain F12 were cultured in RPMI 1640 cell culture medium supplemented with 10% v/v human A+ serum as described (7, 19, 20). Packed human erythrocytes and serum were purchased from the Department of Transfusion Medicine, University Hospital Aachen (Aachen, Germany). The ethics commission of RWTH Aachen University approved all work on human blood. The sera FH concentrations were measured using the MicroVue Factor H EIA kit (Quidel). Parasite synchronization was achieved by sorbitol treatment as described (21). SZ-iRBCs were purified using Percoll gradient centrifugation as described (22). MZs were mechanically purified from enriched mature SZs using a published protocol (23).

A total of 1 × 106 SZ-iRBCs, MZs, or noninfected RBCs (niRBCs) were incubated in cell culture medium containing 50% v/v normal human serum (NHS) or FHR-1–deficient NHS (ΔFHR-1–NHS) for 1 h at 37°C. In some experiments, the sera were supplemented with 30 μg/ml rFHR-1. Following three washes with PBS, the cell pellets were resuspended in 5× SDS sample buffer and heated for 10 min at 94°C. For detection of FH, FHR proteins, and TCCs, gel electrophoreses were conducted under nonreducing conditions with sample buffer lacking DTT and 2-ME. For the detection of C3 products, reducing conditions were applied. Immunoblotting was performed as described (7), using pAb FHR-1, mAb FH, mAb C3, or mAb TCC. Detection of the plasmodial endoplasmic reticulum–resident protein Pf39 (24), using pAb Pf39, served as a loading control. Scanned blots were processed using Adobe Photoshop CS software, and band intensities were measured from three independent Western blot analyses (WBAs) using the ImageJ program, version 1.51j8 (National Institutes of Health). Data analysis was performed using Microsoft Excel 2010 and GraphPad Prism, version 5.01.

A total number of 1 × 106 SZ-iRBCs, MZs, or niRBCs were incubated with cell culture medium containing 50% v/v NHS or ΔFHR-1–NHS for 1 h at 37°C. In some experiments, the sera were supplemented with 30 μg/ml rFHR-1. Following three washes with PBS to remove unbound proteins, the cells were spun down, resuspended in 200 μl of 4% w/v paraformaldehyde/PBS, and subjected to ELISA as described (7), using mAb FH, mAb iC3b, or mAb TCC. Absorbance was measured with the Tecan Spark 10M multimode microplate reader. The experiments were performed in triplicate; data analysis was performed using MS Excel 2010 and GraphPad Prism, version 5.01.

A synchronized culture containing mature SZ-iRBCs with a starting parasitemia of 0.5% was cultivated in cell culture medium containing 10% v/v NHS, ΔFHR-1–NHS, or heat-inactivated serum (HIS), which was inactivated for 1 h at 55°C. In some experiments, the ΔFHR-1–NHS was supplemented with 100 μg/ml rFHR-1. The medium was changed every 12 h to ensure the presence of active complement. Giemsa smears were performed every 12 h postseeding to determine the parasitemia (iRBCs/RBCs total) in 10 optical fields per setting. The experiments were performed in triplicate; data analysis was performed using-MS Excel 2010 and GraphPad Prism version 5.01.

Initially, we compared binding of FHR-1 and FH by the P. falciparum blood stages. Purified MZs, SZ-iRBCs, and niRBCs were incubated in 50% v/v NHS for 1 h. Lysates were subjected to WBA using pAb FHR-1, which revealed that both the intact SZ-iRBCs as well as the MZs bind the two FHR-1 glycosylation variants, FHR-1α (34 kDa) and FHR-1β (36 kDa), in addition to the 155-kDa FH (Fig. 1B, Supplemental Fig. 1A). When tested on NHS, the Ab showed minor cross-reactivity with FHR-2 and its glycosylation variant FHR-2α, as well as with FHR-5. Further, a faint recognition of the FHR proteins in the lysates of niRBCs could be detected, whereas no bands were observed when SZ-iRBCs, MZs, or niRBCs were incubated with PBS (Supplemental Fig. 1A).

FIGURE 1.

FHR-1 competes with FH for binding to P. falciparum blood stages. (A) Domain structures of FH and FHR-1. The binding sites for C3b, including the decay-accelerating (DA) and cofactor (CoF) activity sites as well as the cell surface (CS) recognition sites, are indicated. (B) Binding of FHR-1 to blood stage parasites. SZs, MZs, or niRBCs were incubated with 50% v/v NHS for 1 h, and lysates were subjected to WBA using pAb FHR-1 to detect the glycosylation variants FHR-1β (36 kDa) and FHR-1α (34 kDa) as well as FH (155 kDa). Cross-reactivity with FHR-2 (24 kDa) and the glycosylation variant FHR-2α (29 kDa), as well as FHR-5 (64 kDa), was also detected. NHS was loaded as a positive control. (C) Binding of FH by SZs in the presence of rFHR-1. SZs or niRBCs were incubated with 50% v/v NHS in the absence or presence of 30 μg/ml rFHR-1 for 1 h. Lysates were then subjected to WBA using mAb FH to detect FH in the samples. Detection of Pf39 was used as a loading control. (D) FH binding by SZs in the presence of rFHR-1. SZs or niRBCs were treated as described in (C) and subjected to ELISA, using mAb FH (mean ± SD; niRBCs plus NHS set to 1; compare with Supplemental Fig. 1B). Significant differences are indicated. *p < 0.05, **p < 0.01, Student t test. (E) FH binding by MZs in the presence of rFHR-1. MZs were treated and ELISA was performed as described in (D) (mean ± SD; NHS set to 1; compare with Supplemental Fig. 1C, 1D). *p < 0.05, Student t test. (F) Binding of FH by SZs in the absence of FHR-1. SZs or niRBCs were incubated with 50% v/v NHS or ΔFHR-1–NHS (ΔNHS) for 1 h, and lysates were subjected to WBA using mAb FH to detect FH. Detection of Pf39 was used as a loading control. (G) FH binding by SZs in the absence of FHR-1. SZs or niRBCs were treated as described in (F) and subjected to ELISA using mAb FH. The experiment was performed in triplicate (mean ± SD; niRBC/NHS set to 1; compare with Supplemental Fig. 1F). Significant differences are indicated. ***p < 0.001, Student t test. (H) FH binding by MZs in the absence of FHR-1. MZs were treated and ELISA was performed as described in (G) (mean ± SD; NHS set to 1; compare with Supplemental Fig. 1G, 1H). **p < 0.01, Student t test. (I) Dependence of FH binding by SZs on FHR-1. SZs were incubated with 50% v/v ΔNHS in the absence or presence of 30 μg/ml rFHR-1 for 1 h, and lysates were subjected to WBA using mAb FH to detect FH. Detection of Pf39 was used as a loading control. (J) Dependence of FH binding by SZs on FHR-1. SZs were treated as described in (I) and subjected to ELISA using mAb FH. The experiment was performed in triplicate (mean ± SD; ΔNHS without rFRH-1 set to 1; compare with Supplemental Fig. 1I). **p < 0.01, Student t test. (K) Dependence of FH binding by MZs on FHR-1. MZs were treated and ELISA was performed as described in (J). Significant differences are indicated (compare with Supplemental Fig. 1J, 1K). *p < 0.05, Student t test. The data (D, E, G, H, J, and K) are representative of one of three independent experiments.

FIGURE 1.

FHR-1 competes with FH for binding to P. falciparum blood stages. (A) Domain structures of FH and FHR-1. The binding sites for C3b, including the decay-accelerating (DA) and cofactor (CoF) activity sites as well as the cell surface (CS) recognition sites, are indicated. (B) Binding of FHR-1 to blood stage parasites. SZs, MZs, or niRBCs were incubated with 50% v/v NHS for 1 h, and lysates were subjected to WBA using pAb FHR-1 to detect the glycosylation variants FHR-1β (36 kDa) and FHR-1α (34 kDa) as well as FH (155 kDa). Cross-reactivity with FHR-2 (24 kDa) and the glycosylation variant FHR-2α (29 kDa), as well as FHR-5 (64 kDa), was also detected. NHS was loaded as a positive control. (C) Binding of FH by SZs in the presence of rFHR-1. SZs or niRBCs were incubated with 50% v/v NHS in the absence or presence of 30 μg/ml rFHR-1 for 1 h. Lysates were then subjected to WBA using mAb FH to detect FH in the samples. Detection of Pf39 was used as a loading control. (D) FH binding by SZs in the presence of rFHR-1. SZs or niRBCs were treated as described in (C) and subjected to ELISA, using mAb FH (mean ± SD; niRBCs plus NHS set to 1; compare with Supplemental Fig. 1B). Significant differences are indicated. *p < 0.05, **p < 0.01, Student t test. (E) FH binding by MZs in the presence of rFHR-1. MZs were treated and ELISA was performed as described in (D) (mean ± SD; NHS set to 1; compare with Supplemental Fig. 1C, 1D). *p < 0.05, Student t test. (F) Binding of FH by SZs in the absence of FHR-1. SZs or niRBCs were incubated with 50% v/v NHS or ΔFHR-1–NHS (ΔNHS) for 1 h, and lysates were subjected to WBA using mAb FH to detect FH. Detection of Pf39 was used as a loading control. (G) FH binding by SZs in the absence of FHR-1. SZs or niRBCs were treated as described in (F) and subjected to ELISA using mAb FH. The experiment was performed in triplicate (mean ± SD; niRBC/NHS set to 1; compare with Supplemental Fig. 1F). Significant differences are indicated. ***p < 0.001, Student t test. (H) FH binding by MZs in the absence of FHR-1. MZs were treated and ELISA was performed as described in (G) (mean ± SD; NHS set to 1; compare with Supplemental Fig. 1G, 1H). **p < 0.01, Student t test. (I) Dependence of FH binding by SZs on FHR-1. SZs were incubated with 50% v/v ΔNHS in the absence or presence of 30 μg/ml rFHR-1 for 1 h, and lysates were subjected to WBA using mAb FH to detect FH. Detection of Pf39 was used as a loading control. (J) Dependence of FH binding by SZs on FHR-1. SZs were treated as described in (I) and subjected to ELISA using mAb FH. The experiment was performed in triplicate (mean ± SD; ΔNHS without rFRH-1 set to 1; compare with Supplemental Fig. 1I). **p < 0.01, Student t test. (K) Dependence of FH binding by MZs on FHR-1. MZs were treated and ELISA was performed as described in (J). Significant differences are indicated (compare with Supplemental Fig. 1J, 1K). *p < 0.05, Student t test. The data (D, E, G, H, J, and K) are representative of one of three independent experiments.

Close modal

We then investigated binding of FH by SZ-iRBCs in the presence of rFHR-1. SZ-iRBCs and niRBCs were incubated with 50% v/v NHS in the presence or absence of 30 μg/ml rFHR-1 for 1 h. Lysates were subjected to WBA using mAb FH and displayed decreased detection of FH in the lysates of rFHR-1–incubated SZ-iRBCs compared with parasites cultivated in the absence of rFHR-1 (Fig. 1C). No difference in FH binding, however, was seen in the lysates of niRBCs under either condition. Quantification of FH band intensities (quantitative WBA [qWBA]), as well as ELISA, confirmed a significantly decreased binding of FH by SZ-iRBCs in the presence of rFHR-1 (Fig. 1D, Supplemental Fig. 1B). We also investigated FH binding in the presence of rFHR-1 by MZs via ELISA and qWBA. Again, we observed significantly decreased detection of bound FH when the MZs were incubated with rFHR-1–supplemented NHS (Fig. 1E, Supplemental Fig. 1C, 1D).

We subsequently studied FH binding and complement evasion by blood stage parasites in the absence of FHR-1. For this we used blood from donors with a FHR-1 deficiency. Lack of FHR-1 was confirmed by WBA using pAb FHR-1, and equal FH levels in regular and ΔFHR-1-NHS was also confirmed (Supplemental Fig. 1E). SZ-iRBCs and niRBCs were incubated with 50% v/v NHS or ΔFHR-1–NHS for 1 h. Lysates were immunoblotted with mAb FH to demonstrate increased binding of FH to SZ-iRBCs in the absence of FHR-1 (Fig. 1F). Subsequent ELISA and qWBA confirmed that both SZ-iRBCs and MZs bound significantly more FH when incubated with ΔFHR-1–NHS instead of NHS, whereas no differences in FH binding were observed for niRBCs (Fig. 1G, 1H, Supplemental Fig. 1F–H). This effect could be reversed by addition of rFHR-1. When either SZ-iRBCs or purified MZs were incubated in 50% v/v ΔFHR-1–NHS supplemented with 30 μg/ml rFHR-1, levels of bound FH were significantly lower compared with parasites incubated in ΔFHR-1–NHS without rFHR-1, as demonstrated by ELISA and qWBA, using mAb FH (Fig. 1I–K, Supplemental Fig. 1I–K).

FHR-1 deficiency also impaired C3b inactivation. SZ-iRBCs were incubated with 50% v/v NHS or ΔFHR-1–NHS for 1 h, and lysates were subjected to WBA using pAb C3 to detect C3b and its cleavage products. The 181-kDa protein C3b is composed of the two peptide chains α´ (109 kDa) and β (75 kDa), and its inactivation results in α´ processing and the generation of peptides α´1 and α´2. Immunoblotting detected the α´1 (67 kDa) and α´2 (40 kDa) peptides in both lysates (Fig. 2A). The quantification of the α´1 band intensities by qWBA revealed higher peptide levels in lysates of SZ-iRBCs when these were cultivated in ΔFHR-1–NHS compared with parasites incubated in NHS (Fig. 2B). In contrast, levels of unprocessed C3b decreased under these conditions (Fig. 2A).

FIGURE 2.

FHR-1 impairs FH-mediated complement inactivation and growth of P. falciparum blood stages. (A) Dependence of C3b processing by SZs on FHR-1. SZs were incubated with 50% v/v NHS or ΔFHR-1–NHS (ΔNHS) for 1 h, and the lysates were subjected to WBA using mAb C3 to detect C3b (181 kDa), its unprocessed α´ and β chains (109 and 75 kDa, respectively), and the processed α´1 and α´2 peptides (67 and 40 kDa, respectively). (B) Dependence of C3b processing by SZs on FHR-1. SZs were treated as described in (A) and subjected to qWBA using mAb C3. Relative band intensities for the iC3b α´ chain were measured from three independent WBAs (mean ± SD; NHS set to 1). Significant differences are indicated. *p < 0.05, Student t test. (C) Dependence of C3b inactivation by SZs on FHR-1. SZs were incubated with 50% v/v ΔNHS in increasing concentrations of rFHR-1 for 1 h. The generation of iC3b on the SZ surfaces was determined via ELISA using mAb iC3b. The experiment was performed in triplicate (mean ± SD; NHS without rFRH-1 set to 1). Significant differences are indicated. ***p < 0.001, Student t test. (D) Dependence of C3b inactivation by MZs on FHR-1. MZs were treated and ELISA was performed as described in (C). **p < 0.01, Student t test. (E) Dependence of TCC formation in SZs on FHR-1. SZs were incubated with 50% v/v NHS or ΔNHS for 1 h, and lysates were subjected to WBA using mAb TCC to detect TCC (∼330 kDa). (F) Dependence of TCC levels in SZs on FHR-1. SZs were treated as described in (E) and subjected to ELISA using mAb TCC. The experiment was performed in triplicate (mean ± SD; NHS set to 1; compare with Supplemental Fig. 1L). Significant differences are indicated. *p < 0.05, Student t test. (G) Effect of FHR-1 deficiency on parasite growth. Mature SZs with a starting parasitemia of 0.5% were cultured in 10% v/v NHS, ΔNHS, or HIS at 37°C for 48 h. The parasitemia was evaluated every 12 h via Giemsa smears. The experiment was performed in triplicate (mean ± SD). Significant differences in parasitemia are indicated. *p < 0.05, Student t test. (H) Effect of FHR-1 supplementation on parasite growth. Mature SZs were seeded in 10% v/v NHS, HIS, or ΔNHS as described in (G) in the absence or presence of 100 μg/ml rFHR-1. The parasitemia was evaluated at 72 h postseeding. The experiment was performed in triplicate (mean ± SD; HIS set to 100%). Significant differences in parasitemia are indicated. *p < 0.05, Student t test. The data (C, D, and F–H) are representative of one of three independent experiments.

FIGURE 2.

FHR-1 impairs FH-mediated complement inactivation and growth of P. falciparum blood stages. (A) Dependence of C3b processing by SZs on FHR-1. SZs were incubated with 50% v/v NHS or ΔFHR-1–NHS (ΔNHS) for 1 h, and the lysates were subjected to WBA using mAb C3 to detect C3b (181 kDa), its unprocessed α´ and β chains (109 and 75 kDa, respectively), and the processed α´1 and α´2 peptides (67 and 40 kDa, respectively). (B) Dependence of C3b processing by SZs on FHR-1. SZs were treated as described in (A) and subjected to qWBA using mAb C3. Relative band intensities for the iC3b α´ chain were measured from three independent WBAs (mean ± SD; NHS set to 1). Significant differences are indicated. *p < 0.05, Student t test. (C) Dependence of C3b inactivation by SZs on FHR-1. SZs were incubated with 50% v/v ΔNHS in increasing concentrations of rFHR-1 for 1 h. The generation of iC3b on the SZ surfaces was determined via ELISA using mAb iC3b. The experiment was performed in triplicate (mean ± SD; NHS without rFRH-1 set to 1). Significant differences are indicated. ***p < 0.001, Student t test. (D) Dependence of C3b inactivation by MZs on FHR-1. MZs were treated and ELISA was performed as described in (C). **p < 0.01, Student t test. (E) Dependence of TCC formation in SZs on FHR-1. SZs were incubated with 50% v/v NHS or ΔNHS for 1 h, and lysates were subjected to WBA using mAb TCC to detect TCC (∼330 kDa). (F) Dependence of TCC levels in SZs on FHR-1. SZs were treated as described in (E) and subjected to ELISA using mAb TCC. The experiment was performed in triplicate (mean ± SD; NHS set to 1; compare with Supplemental Fig. 1L). Significant differences are indicated. *p < 0.05, Student t test. (G) Effect of FHR-1 deficiency on parasite growth. Mature SZs with a starting parasitemia of 0.5% were cultured in 10% v/v NHS, ΔNHS, or HIS at 37°C for 48 h. The parasitemia was evaluated every 12 h via Giemsa smears. The experiment was performed in triplicate (mean ± SD). Significant differences in parasitemia are indicated. *p < 0.05, Student t test. (H) Effect of FHR-1 supplementation on parasite growth. Mature SZs were seeded in 10% v/v NHS, HIS, or ΔNHS as described in (G) in the absence or presence of 100 μg/ml rFHR-1. The parasitemia was evaluated at 72 h postseeding. The experiment was performed in triplicate (mean ± SD; HIS set to 100%). Significant differences in parasitemia are indicated. *p < 0.05, Student t test. The data (C, D, and F–H) are representative of one of three independent experiments.

Close modal

The beneficial effect of FHR-1 deficiency on C3b inactivation could be reversed by addition of rFHR-1. SZ-iRBCs and MZs were incubated with 50% v/v ΔFHR-1–NHS supplemented with different concentrations of rFHR-1 for 1 h and then analyzed by ELISA using mAb iC3b, which detects a C3b epitope only exposed following FH-mediated C3b cleavage by factor I. ELISA demonstrated significantly decreasing iC3b levels for SZ-iRBCs and MZs when the rFHR-1 concentrations increased to up to 100 μg/ml (Fig. 2C, 2D). FHR-1 deficiency also affected TCC formation. The 330-kDa TCCs were detectable in lysates of SZ-iRBCs incubated with either 50% v/v NHS or ΔFHR-1–NHS when WBA with the mAb TCC was performed (Fig. 2E). TCC levels, however, were significantly decreased in SZ-iRBCs incubated in ΔFHR-1–NHS compared with those cultured with NHS, as demonstrated by ELISA and qWBA (Fig. 2F, Supplemental Fig. 1L).

In a final set of experiments, we investigated how FHR-1 deficiency affects parasite growth in vitro. Mature SZs with a starting parasitemia of 0.5% were cultured in 10% v/v NHS, ΔFHR-1–NHS, or HIS over a period of 48 h. Although there were no significant differences seen at 12 h postseeding, in the following 36 h (thus, during the second generation cycle), the numbers of iRBCs significantly decreased when the parasites were cultivated in NHS. In contrast, when cultured in ΔFHR-1–NHS, the iRBC numbers slightly decreased but did not differ significantly from those kept in HIS (Fig. 2G). The growth-promoting effect of ΔFHR-1–NHS could be reversed by addition of rFHR-1. When parasites were incubated in ΔFHR-1–NHS for 72 h in the presence of 100 μg/ml FHR-1, the parasitemia significantly decreased and was comparable to cultures kept in NHS (Fig. 2H). The highest parasitemia was observed under HIS conditions.

Our combined data demonstrate that FHR-1 competes with FH for binding to SZ-iRBCs and free MZs, consequently impairing C3b inactivation and promoting TCC formation on the SZ-iRBC surface. This reduced regulatory activity of the alternative complement pathway eventually results in impaired complement evasion by the P. falciparum blood stages and, thus, in parasite killing, indicating that FHR-1 is a crucial regulator of malaria infection.

To date, not much is known about the regulatory function of FHR-1. It is the most abundant FHR protein, with an approximate plasma concentration of 70–100 μg/ml (25). Its homology with FH suggests related functions, including binding to C3b and heparin and, thus, recognition of cell surfaces. Consistent with the lack of domains homologous to the complement regulatory region of FH, however, no cofactor and convertase decay-accelerating activities were observed for FHR-1 (17, 26, 27). Previous studies suggested that FHR-1 binds to the surfaces of microbes that otherwise bind FH, including Borrelia burgdorferi, Neisseria gonorrheae, Staphylococcus aureus, and Candida albicans, to modulate innate immunity by enhancing complement activity (2833). Because the FH domains conserved among the FHR proteins are those that mediate the binding to microbial proteins, it was recently postulated that the FHR proteins have evolved as decoys to reduce the amount of FH acquired by the pathogens, consequently promoting their elimination by the human complement system (11, 34, 35).

Consistent with these reports, the results of our study let us conclude that FHR-1 competes with FH for the plasmodial FH receptors, thereby limiting the regulatory activity of FH during complement evasion of the P. falciparum blood stages. Hence, we postulate that FHR-1 functions as a protective complement factor to reduce plasmodial virulence. Future studies will need to identify the precise molecular mechanisms by which FHR-1 modulates human immunity against microbial infections and to investigate the potential susceptibility of individuals harboring homozygous FHR-3/FHR-1 gene deletions to these infections.

This work was supported by Grants PR905/12-1 (to G.P.) and SK46/4-1 (to C.S.) from the Deutsche Forschungsgemeinschaft. T.F.d.A.R. received a fellowship from the Science without Borders Foundation Programme CAPES. G.P. is the recipient of a Heisenberg professorship from the Deutsche Forschungsgemeinschaft.

The online version of this article contains supplemental material.

Abbreviations used in this article:

CCP

complement control protein domain

FH

factor H

FHR

FH-related

ΔFHR-1–NHS

FHR-1–deficient NHS

HIS

heat-inactivated serum

iC3b

inactivated C3b

iRBC

infected RBC

MZ

merozoite

NHS

normal human serum

niRBC

noninfected RBC

pAb

polyclonal Ab

qWBA

quantitative WBA

rFHR-1

recombinant FHR-1

SZ

schizont

SZ-iRBC

SZ-infected RBC

TCC

terminal complement complex

WBA

Western blot analysis.

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The authors have no financial conflicts of interest.

Supplementary data