Molecular analysis of erythrocyte invasion in Plasmodium falciparum isolates from Senegal

Abstract

The human malaria parasite, Plasmodium falciparum, utilizes multiple ligand-receptor interactions for the invasion of human erythrocytes. Members of the reticulocyte binding protein homolog (PfRh) family have been shown to be critical for directing parasites to alternative erythrocyte receptors that define invasion pathways. Recent studies have identified gene amplification, sequence polymorphism, and variant expression of PfRh paralogs as mechanisms underlying discrimination between pathways for invasion. In this study, we find considerable heterogeneity in the invasion profiles of clonal, uncultured P. falciparum parasite isolates from a low-transmission area in Senegal. Molecular analyses revealed minimal variation in protein expression levels of the PfRh ligands, PfRh1, PfRh2a, and PfRh2b, and an absence of gene amplification in these isolates. However, significant sequence polymorphism was found within repeat regions of PfRh1, PfRh2a, and PfRh2b. Furthermore, we identified a large sequence deletion ( approximately 0.58 kb) in the C-terminal region of the PfRh2b gene at a high prevalence in this population. In contrast to findings of earlier studies, we found no associations between specific sequence variants and distinct invasion pathways. Overall these data highlight the importance of region-specific elaborations in PfRh sequence and expression polymorphisms, which has important implications in our understanding of how the malaria parasite responds to polymorphisms in erythrocyte receptors and/or evades the immune system.

FIG. 1.

Plasmodium falciparum strains from Dakar, Senegal, routinely use alternate erythrocyte invasion pathways. (A) Efficiency of invasion by P. falciparum of neuraminidase (Nm)-treated erythrocytes, chymotrypsin (Chymo)-treated erythrocytes, and trypsin (Tryp)-treated erythrocytes. (B) Lack of significant correlation of efficiencies of invasion of enzyme-treated erythrocytes for the series of isolates. Rs and P values were derived by Spearman's rank correlation.

FIG. 2.

P. falciparum isolates from Senegal exhibit minimal variation in the expression of PfRh1, PfRh2a, and PfRh2b. Western blots of supernatant extracts probed with antisera specific for PfRh2a, PfRh2b, PfRh1, or PfSera5 as a loading control. The various sizes of PfRh2a and PfRh2b in the different isolates result from both sequence polymorphisms in the repetitive regions of these proteins and proteolytic breakdown products that are commonly observed even in laboratory isolates. Additionally, some isolates contain a large sequence deletion in PfRh2b (indicated by an arrow).

FIG. 3.

Lack of PfRh1 amplification in Senegalese field isolates. Southern blot to determine PfRh1 copy number. Pfdhps is a single-copy gene used as a control. PfRh1 copy number was determined for each sample by comparing the Pfdhps/PfRh1 signal ratio for each Senegalese isolate to that for HB3, which is known to have a PfRh1 copy number of 1.

FIG. 4.

Identification of a large sequence polymorphism within the C terminus unique region of PfRh2b in Senegalese isolates. (A) Schematic of PfRh2a and PfRh2b showing the 3′ repetitive, unique, transmembrane, and cytoplasmic tail regions. Primers used for the PCR mapping of the deletion are indicated. (B) PCR mapping identifies a major sequence deletion in the unique region of PfRh2b in representative Senegalese isolates. (C) Southern blot of the C-terminal region of PfRh2b, confirming the presence of the deletion in the chromosomal DNA in a subset of Senegalese isolates. A fragment of the unique region upstream of the deletion, amplified with P1 and P3, was used as a probe. (D) The sequence of the PfRh2b deletion is identical in multiple Senegalese isolates and the Thai isolate T996w (T996 obtained from WEHI, Melbourne). Alignment of the unique region from Senegalese isolate with and without the deletion with those of HB3 and T996w.

FIG. 5.

The role of the PfRh2bdel polymorphism in the Senegalese isolates. (A) Association between invasion pathway utilization and the presence of the PfRh2bdel polymorphism. P value indicated was derived using a Mann-Whitney U test. DEL, deletion present; FULL, full-length sequence; NS, not significant. (B) PCR assay for the identification of polymorphisms in the downstream deletion in PfRh2b. (C) High prevalence of PfRh2b deletion in Senegalese population, determined using the PCR-based assay for the detection of the PfRh2bdel in isolates from Pikine (n = 23) or Thies (n = 19).

FIG. 6.

Extensive and novel polymorphisms in the repetitive regions of PfRh1, PfRh2a, and PfRh2b. (A) Alignment of PfRh1 repetitive sequence polymorphisms and their allelic designations according to the convention of Lobo et al. (17) (amino acids 2734 to 2741 according to the 3D7 sequence, accession no. AAQ10315). Isolates that contain polymorphisms previously described by Lobo et al. (17): 3D7-LIKE (P18, T14, and T15); FFS-LIKE (P04, P06, T04, T09, and T10); Dd2-LIKE (T22). Isolates that contain novel polymorphisms: NOVEL A (T11, T18, P02, P12, and T13); NOVEL B (P15), and NOVEL C (P09). (B) Alignment of sequence polymorphisms in the repetitive regions of PfRh2a in multiple Senegalese isolates. Polymorphism in the repetitive regions and the “pep” polymorphisms (amino acids 2734 to 2741 according to the FVO sequence, accession no. AAN39446) are indicated in bold. Isolates that contain polymorphisms previously described by Lobo et al. (17), FVO-LIKE (P04, P05, P06, P12, P41, P43, T09, T10, T11, and T13). Isolates that contain novel polymorphisms, NOVEL A (P15 and T15), NOVEL B (P18), and NOVEL C (T14). (C) Alignment of sequence polymorphisms in the repetitive regions of PfRh2b in multiple Senegalese isolates. The repetitive region and “pep” polymorphisms (amino acids 2769 to 2777 according to the 3D7 sequence, accession no. AAN39447) are indicated in bold. Isolates that contain polymorphisms previously described by Lobo et al. (17): 3D7-LIKE (P04,P05,P41,P43,T09,T10,T12,T13,T14,T18) and GVM-LIKE (P06,P15,P18,T22). Isolates that contain novel polymorphisms: NOVEL A (P09), NOVEL B (T11), and NOVEL C (P12). Isolates containing the downstream PfRh2bdel polymorphism are shown in bold.