Single amino acid substitution in Plasmodium yoelii erythrocyte ligand determines its localization and controls parasite virulence

Abstract

The major virulence determinant of the rodent malaria parasite, Plasmodium yoelii, has remained unresolved since the discovery of the lethal line in the 1970s. Because virulence in this parasite correlates with the ability to invade different types of erythrocytes, we evaluated the potential role of the parasite erythrocyte binding ligand, PyEBL. We found 1 amino acid substitution in a domain responsible for intracellular trafficking between the lethal and nonlethal parasite lines and, furthermore, that the intracellular localization of PyEBL was distinct between these lines. Genetic modification showed that this substitution was responsible not only for PyEBL localization but also the erythrocyte-type invasion preference of the parasite and subsequently its virulence in mice. This previously unrecognized mechanism for altering an invasion phenotype indicates that subtle alterations of a malaria parasite ligand can dramatically affect host-pathogen interactions and malaria virulence.dense granule ͉ invasion ͉ malaria ͉ microneme ͉ transfection T he rodent malaria parasite Plasmodium yoelii yoelii has been widely studied to understand the interactions between the malaria parasite and the host cell (1). The nonlethal 17X line mainly infects young erythrocytes (reticulocytes), whereas the lethal 17XL and YM lines infect a wide range of erythrocytes. These lines have previously been studied to identify the genetic determinants of virulence (2, 3). These differences in erythrocyte invasion preference suggest the possible involvement of a parasite ligand that recognizes erythrocyte surface receptors; however, the actual molecular basis of the observed invasion preference differences remains unclear.Erythrocyte invasion by the malaria merozoite is a multistep process, initiated by reversible binding to the erythrocyte surface, followed by the establishment of a tight junction between the apical end of the merozoite and erythrocyte surface and the subsequent movement of the merozoite into the nascent parasitophorous vacuole. Each step involves specific interactions between parasite ligands and erythrocyte receptors. Among the ligands of malaria parasites, the best characterized is a type I integral transmembrane protein encoded by the ebl (erythrocytebinding-like) gene family. Upon release from the micronemes, EBL proteins recognize erythrocyte receptors and initiate the formation of the tight junction. The importance of EBL in malaria virulence is exemplified in the human malaria parasite Plasmodium vivax, which uses an EBL orthologue, PvDBP, to recognize the Duffy antigen on the erythrocyte surface. Because the parasite is apparently unable to use an alternative invasion pathway, individuals in whom the Duffy antigen is not expressed on the erythrocyte surface are completely resistant to P. vivax (4,5). Because of this dramatic association between the disruption of a host-pathogen interaction and protection against a malaria parasite, PvDBP and the Plasmodium falciparum EBL orthologue, EBA-175, have been targeted for ...

Gene encoding erythrocyte binding ligand linked to blood stage multiplication rate phenotype inPlasmodium yoelii yoelii

“…Moreover, sequence analysis of pyebl in fast-and slow-multiplying lines of P. y. yoelii revealed a SNP at position 2340 in pyebl that associates with the multiplication rate phenotypes. This is consistent with the findings of Otsuki and others (14) in suggesting the involvement of pyebl in determining multiplication rate phenotype in these malaria parasites. Our studies also indicated effects of at least 1 additional locus on multiplication rate, as did the findings of Otsuki and colleagues on virulence in P. y. yoelii.…”

“…Furthermore, sequencing the pyebl gene revealed a SNP at position 2340 that distinguished all of the slow multiplying lines, which were identical at this position, from the fast multiplying line. This SNP leads to an amino acid substitution in region 6 of the PyEBL protein and is the same SNP as was identified as controlling the virulence phenotype differences between 17X and 17XYM by Otsuki and colleagues (14). Together, these results strongly imply that the SNP at position 2340 of pyebl is the major genetic determinant of both the multiplication rate and virulence difference phenotypes in the lines of P. y. yoelii that have been investigated in our two studies.…”

“…These circumstances and this hypothesis are consistent with the above analysis indicating the involvement of a single major genetic locus. However, in their replacement transfection experiments, Otsuki and colleagues found that, although the pyebl gene constituted a major determinant of the multiplication rate phenotype, the influence of other genes was also likely (14). Moreover, although our LGS analyses, which involved genomewide coverage, found no direct evidence of strong multiplication rate selection outside the region containing the pyebl gene on P. yoelii chromosome 13, two other AFLP markers (33XC CA05TT 17XYM and 33XC GT02TT 17XYM) were, nevertheless, found to be under strong selection after the second and third rounds of infection of the uncloned cross progeny in mice.…”

“…These findings are consistent with position 2340 of pyebl being a genetic determinant of the multiplication rate phenotype in this parasite. Position 2340 of pyebl is the mutation shown by Otsuki and colleagues (14), in their transfection experiments, to determine virulence in these parasites.…”

“…Our initial LGS analyses of genetic crosses between the fast-(17XYM) and slowly multiplying (33XC) lines of P. y. yoelii indicated that a region in the parasite's genome, spanning about half a megabase on P. y. yoelii chromosome 13, contained a major gene or genes that control fast or slow multiplication of the parasites in the blood. While this work was in progress we learned from Otsuki and colleagues (14) that replacement transfection of the P. y. yoelii 17XYM gene encoding the erythrocyte binding ligand (pyebl) conferred the fast multiplication phenotype on a wild-type line of P. y. yoelii. Following this communication, we located pyebl in the P. y. yoelii genome within the region on chromosome 13 identified by LGS analysis to determine multiplication rate.…”