Our findings link polymorphisms in PfRH5, a newly identified erythrocyte binding protein, to switches of host receptor recognition by P. falciparum malaria parasites. In a genetic cross of two parasites that differ dramatically in their ability to infect A. nancymaae monkeys (7G8, GB4), inheritance of the PfRH5 allele from one parent or the other in the haploid progeny proved to be the dominant determinant of infectivity. Transfection and allelic exchange results show that non-synonymous changes in just two codons were sufficient to convert the non-invasive parent (7G8) to a parasite (7G8KV, carrying the equivalent of the GB4 parent allele) able to invade A. nancymaae erythrocytes and infect a monkey in vivo. Of these two polymorphisms, codon change I204K accounted for nearly all of the conversion in invasion phenotype. Furthermore, the invasion of A. nancymaae erythrocytes correlated completely with binding of a PfRH5 Mr 28 K fragment from parasites of the cross.
QTL analysis did not identify loci other than PfRH5 that could be linked to A. nancymaae infectivity in 7G8×GB4 progeny recovered from the chimpanzee host. However, several observations suggest that additional genetic determinants may modulate the PfRH5 pathway for A. nancymaae erythrocyte invasion and infection of A. nancymaae monkeys, as: (1) different 7G8×GB4 progeny required less than 10 days to more than 50 days to produce patent A. nancymaae infections; (2) progeny KB8, JC3 and XF12 produced no infections in A. nancymaae even though these clones inherited the GB4 allele of PfRH5 and invaded monkey erythrocytes well in vitro; (3) replacement of the 7G8 PfRH5 allele by the GB4 allele in the JB12 progeny clone resulted in an A. nancymaae erythrocyte invasion rate about two-thirds of that observed for the same replacement in the 7G8 parent; and (4) non-invasive parasites of the unrelated P. falciparum 3D7 clone remained unable to invade A. nancymaae erythrocytes after they were modified to express PfRH5 polymorphisms of the GB4 allele (3D7KV line). Detection of additional determinants involved in these variations may have been obscured by their multiplicity, interactions with partners, or variable effects among the different inherited genetic backgrounds of the 32 recombinant progeny in this study.
The region of highly skewed inheritance on chromosome 7 contains PfEBA175
, the gene encoding a 175 kDa parasite protein that binds sialic acid residues of erythrocyte surface glycophorin A (Camus and Hadley, 1985
; Sim et al., 1994
). All 32 independent recombinants from the chimpanzee host inherited the PfEBA175
allele and nearby genes on chromosome 7 from the 7G8 rather than the GB4 parent. In PfEBA175, the erythrocyte binding domain is defined by a 616 amino acid region (Sim et al., 1994
), which differs in the 7G8 and GB4 parasites by nine amino acid residues at polymorphic sites reported previously (data not shown; Liang and Sim, 1997
). As the PfEBA175
gene is proposed to be a determinant of primate specificity (Martin et al., 2005
), it is possible that inheritance of the 7G8 allele vs
. the GB4 allele reflects upon the function of the PfEBA175 protein in primate sialic acid binding.
PfRH5 is the smallest member of the RH family. Its 63 kDa amino acid sequence aligns most closely with that of PrRH5, followed by the N-terminal regions of the 370 kDa PfRH2a and 383 kDa PfRH2b proteins (Rayner et al., 2000
). PfRH5 therefore lacks regions corresponding to more than 300 kDa of peptide sequence in the body and C-terminal regions of PfRH2a and PfRH2b, including a predicted transmembrane domain characteristic of other family members. These alignments may help to define binding domains of PfRH2a, PfRH2b and other RH proteins. Our attempts and those of others to disrupt the PfRH5
gene have been unsuccessful (data not shown; Cowman and Crabb, 2006
), in marked contrast to the experimental knock-outs achieved for other members of the RH family as well as various P. falciparum EBA
genes (Cowman and Crabb, 2006
may therefore have a vital role in the ability of merozoites to recognize and enter various human and non-human erythrocytes. The location of PfRH5 at the apical end of the merozoite presumably makes the protein readily available for processing, discharge and binding with host cell proteins that function in the invasion of erythrocytes.
The ability of parasites in the 7G8×GB4 cross to infect A. nancymaae correlates fully with the binding of a Mr 28K fragment from PfRH5 molecules containing a positively charged lysine residue at position 204. Binding of this fragment may reflect a ligand-receptor function of PfRH5 or its processed forms for parasite entry into the A. nancymaae cells. Neuraminidase removal of negatively charged sialic acid residues from the surface of the monkey erythrocytes completely abrogated binding of the Mr 28K fragment and blocked invasion of the parasites in this work. These results suggest that an ionic bond between the positively charged lysine and a negatively charged sialic acid may promote an important ligand-receptor interaction.
Our results also show that human erythrocytes treated with neuraminidase to remove sialic acid residues can bind the Mr
28K fragment of PfRH5 when it contains I204 but not when it contains K204. Non-treated human erythrocytes, however, can bind both forms of the fragment. We note that different polymorphisms are also present in the PfRH5 sequences of other P. falciparum
lines that are able to infect A. nancymaae
erythrocytes (), and many of these polymorphisms involve amino acid class changes, including an I204R substitution in the Palo Alto PfRH5 that may have a functional effect similar to the I204K substitution in GB4. Considering the importance of sialic acid residues to the binding of PfRH5 domains containing K204 but not I204, we suggest that PfRH5 polymorphisms may enable P. falciparum
parasites to switch their ligand-receptor interactions and adapt to different erythrocyte surface molecules. Point mutations in the sequences of two other P. falciparum
ligands, JESEBL/EBA181 and BAEBL/EBA140, also alter their molecular specificity for different receptors on erythrocytes (Mayer et al., 2002
; Mayer et al., 2004
). These and other polymorphisms provide an important adaptive ability in the various pathways P. falciparum
uses to infect different human erythrocytes. The utility of these pathways is severely restricted in the non-natural host cells of A. nancymaae
, accounting for the dependence of LC12 and other 7G8×GB4 parasites on PfRH5 as a single ligand for invasion.
Parasites that infect across species barriers are reported from a number of primate malarias. For example, P. knowlesi
parasites that naturally infect monkeys have produced outbreaks of malaria in human populations (Cox-Singh et al., 2008
). The close relationship between macaque P. cynomolgi
and human P. vivax
parasites (Cornejo and Escalante, 2006
) points to the existence of shared reservoirs of infection before a recent evolutionary divergence, and the near identity of P. simium
to P. vivax
may reflect an even more recent host switch back to monkeys from humans (Cornejo and Escalante, 2006
). In the present case, there is no evidence that Aotus
monkeys carry a reservoir of P. falciparum
parasites in South America (Collins, 1994
). Some of the P. falciparum
strains able to infect A. nancymaae
in our study were isolated in Africa (GB4, Ghana; Palo Alto, Uganda) where these parasites had not been under selective pressure to adapt to Aotus
monkeys. It seems likely that the ability of these parasites to invade A. nancymaae
erythrocytes derives from the diversity of ligand-receptor interactions exploited by P. falciparum
in its various pathways to invade human erythrocytes.