Erythrocyte binding protein PfRH5 polymorphisms determine species-specific pathways of Plasmodium falciparum invasion

Abstract

Some human malaria Plasmodium falciparum parasites, but not others, also cause disease in Aotus monkeys. To identify the basis for this variation, we crossed two clones that differ in Aotus nancymaae virulence and mapped inherited traits of infectivity to erythrocyte invasion by linkage analysis. A major pathway of invasion was linked to polymorphisms in a putative erythrocyte binding protein, PfRH5, found in the apical region of merozoites. Polymorphisms of PfRH5 from the A. nancymaae-virulent parent transformed the nonvirulent parent to a virulent parasite. Conversely, replacements that removed these polymorphisms from PfRH5 converted a virulent progeny clone to a nonvirulent parasite. Further, a proteolytic fragment of PfRH5 from the infective parasites bound to A. nancymaae erythrocytes. Our results also suggest that PfRH5 is a parasite ligand for human infection, and that amino acid substitutions can cause its binding domain to recognize different human erythrocyte surface receptors.

FIGURE 1. P. falciparum parasite infectivities to Aotus monkeys and to their erythrocytes in vitro

A) Parasitemias obtained in four A. nancymaae monkeys after intravenous inoculation of 1×10⁷ GB4-parasitized erythrocytes. All infections were cured with 25 mg/kg mefloquine (arrow). B) A. nancymaae erythrocyte invasion rates by the parents and 32 independent recombinant progeny from the P. falciparum 7G8×GB4 cross. Percentage values are relative to rates of invasion into human control erythrocytes ± SEM. C) Infectivity of 10 7G8×GB4 progeny clones to A. nancymaae monkeys. All infections were cured with 25 mg/kg mefloquine (arrows).

FIGURE 2. Identification of candidate genes for the A. nancymaae invasion phenotype

A) Genome-wide plot of the ratios of 7G8:GB4 markers among the 32 independent recombinants. A peak containing PfEBA175 (arrow) identifies a region of chromosome 7 that is severely skewed in favor of the 7G8 parent. A dashed line indicates the 0.5 ratio expected from random assortment. B) Logarithm of odds (LOD) scores from the primary scan for quantitative trait loci (QTL) associated with A. nancymaae erythrocyte invasion. The peak with LOD of 11.8 maps to the sub-telomeric region of chromosome 4. A dashed line represents the significance threshold (0.05). C) Map of the chromosome region linked to A. nancymaae invasion phenotype. A. nancymaae erythrocyte invasion phenotypes (%’s in brackets) are shown for five progeny clones carrying different chromosome segments from each of the parents in the mapped region (chr 4, chromosome 4; solid black segments, GB4; open segments, 7G8). The Aotus invasion locus maps to a 13.7 kb sequence defined by the microsatellite marker C4M30 and a single nucleotide polymorphism (SNP) in the pseudogene PEBL. The exons and transcription directions of the PfRH4 and PfRH5 genes are indicated by connected gray bars and arrows, respectively, while the reading frame direction of the pseudogene PEBL is shown with a dashed arrow. Comparisons of the genomic and cDNA sequences of the 7G8 PfRH5 gene, including 5'- and 3'-rapid amplification of cDNA ends (RACE) sequencing, confirmed the two exon structure documented in PlasmoDB (www.plasmodb.org).

FIGURE 3. Schematic representation and sequence alignment of PfRH5 with members of the RH superfamily

A) Organization of PfRH5 and PfRH2b. ss, putative signal sequence (gray box); tm, transmembrane region (black box). PfRH5 has 526 predicted amino acids and an estimated molecular weight of 63 kDa. B) Alignments of predicted RH5 and homologous N-terminal RH sequences from P. falciparum, P. reichenowi and P. yoelii. Conserved residues are shaded black and semi-conserved regions shaded grey. PfRH5 carries cysteine residues (purple highlight) at four of the five positions where there are conserved or semi-conserved cysteines in the other sequences (185, 224, 317, 345, 351). Amino acids 1–24 of PfRH5 constitute a possible signal peptide (SignalP 3.0 program; Bendtsen et al., 2004). Asterisks mark positions 204 and 407 where polymorphisms occur between the 7G8 and GB4 clones; positions of differences in other isolates (Table 1) are marked with closed circles. Lysine occurs in position 204 of PfRH5 in GB4, but not in other P. falciparum lines of our survey (Table 1); this residue is also encoded at the equivalent codon position in the P. reichenowi PrRH5 sequence and may represent a persisting ancestral codon. Amino acids of the synthetic MAP4 peptide are overlined. Sequences: PfRH5, (7G8 clone, accession number EU433391), PfRH2b (amino acids 1–375, AF312917), PrRH2b (1–473, Y166677), PrRH5 (EF142858), Py235 (partial sequence, 1–464, XM_719883); Pf, P. falciparum; Pr, P. reichenowi, Py, P. yoelii. Sequence homology searches were performed using tblastn and alignments were generated with T-Coffee software (Notredame et al., 2000).

FIGURE 4. Allelic modification of PfRH5 and confirmation of its role in erythrocyte invasion

A) Schematic of the strategy to generate a 7G8^KV parasite by homologous recombination. A portion of the GB4 PfRH5 gene containing the K204 and V407 codons along with 0.2 kb of 3’ flanking sequence replaces its counterpart in the expressed endogenous allele. The hDHFR (human dihydrofolate reductase) drug selection marker is located downstream of the modified PfRH5 allele. Abbreviations: 3’, 3’ UTR of PfRH5; hrp3 5’, promoter region from P. falciparum histidine-rich protein 3; h3’, 3’ UTR of P. falciparum histidine-rich protein 2; Nhe, Nhe I; Not, Not I; Pst, Pst I. B) Southern blot verification of the 11.3 kb Nhe I band resulting from homologous integration in the PfRH5 locus. Genomic DNAs were isolated from untransformed 7G8 parasites and from transformed 7G8^KV parasites containing the GB4 PfRH5 allele. C) Invasion rates of untransformed and transformed P. falciparum lines into A. nancymaae erythrocytes ± SEM. D) Results in three naïve A. nancymaae monkeys after intravenous inoculation of bloodstream forms of GB4 (blue line), 7G8 (green line) or PfRH5-modified 7G8KV parasites (red line). All animals were splenectomized prior to infection and cured with 25 mg/kg mefloquine (arrows).

FIGURE 5. Immunoblot detection of PfRH5 and binding of a Mr 28K fragment to human and A. nancymaae erythrocytes

A) Rabbit antisera raised against a 16 amino acid peptide recognize 16 and 28 kDa fragments of PfRH5 in Escherichia coli. Polypeptides of the appropriate size are present in induced (+) but not uninduced (−) cells. B) Immunoblot analysis of 7G8 parasite extracts from synchronized ring stages (R), early trophozoites (T) and mature schizonts (S). A M_r 67K band is evident in the 7G8 schizont preparation probed by anti-PfRH5 peptide antisera. The signals from repeat probing of the blot with antibodies against human spectrin, an abundant red cell protein present in parasite extracts, verify that protein from comparable numbers of cells was present in each lane. C) The M_r 28K PfRH5 fragment of LC12 parasites binds to A. nancymaae as well as human erythrocytes, consistent with the ability of LC12 to infect both types of cells, while the PfRH5 fragment from 7G8 parent parasites only binds human erythrocytes. D) The M_r 28K PfRH5 fragment from parasites transformed to express K204 (LC12^KV, LC12^KI, 7G8^KV), but not I204 (LC12^II, 7G8^II, 7G8^IV) binds to A. nancymaae erythrocytes. Human erythrocytes bind the PfRH5 fragment from all parasites. E) The human erythrocyte receptor for the PfRH5 fragment of 7G8 parasites is neuraminidase-, trypsin-and chymotrypsin-resistant, while both the human and A. nancymaae erythrocyte receptor for the PfRH5 fragment of LC12 parasites is trypsin- and chymotrypsin-resistant, but neuraminidase-sensitive. U, untreated; N, neuraminidase; T, trypsin; C_T, chymotrypsin.

Figure 6. PfRH5 localization in schizonts and merozoites

A) Immunoconfocal microscopy showing the localization of PfRH5 relative to PfEBA175, a microneme marker. Signals from mature segmented schizonts are shown in the top panes; signals from free merozoites are shown in the bottom panes. B) Immunoconfocal microscopy showing the localization of PfRH5 relative to PfRAP1, a rhoptry marker. Signals from mature schizonts are shown in the top panes; signals from free merozoites are shown in the bottom panes. Scale bar, 2 µm.