Plasmodium falciparum field isolates from South America use an atypical red blood cell invasion pathway associated with invasion ligand polymorphisms

Abstract

Studies of Plasmodium falciparum invasion pathways in field isolates have been limited. Red blood cell (RBC) invasion is a complex process involving two invasion protein families; Erythrocyte Binding-Like (EBL) and the Reticulocyte Binding-Like (PfRh) proteins, which are polymorphic and not fully characterized in field isolates. To determine the various P. falciparum invasion pathways used by parasite isolates from South America, we studied the invasion phenotypes in three regions: Colombia, Peru and Brazil. Additionally, polymorphisms in three members of the EBL (EBA-181, EBA-175 and EBL-1) and five members of the PfRh (PfRh1, PfRh2a, PfRh2b, PfRh4, PfRh5) families were determined. We found that most P. falciparum field isolates from Colombia and Peru invade RBCs through an atypical invasion pathway phenotypically characterized as resistant to all enzyme treatments (NrTrCr). Moreover, the invasion pathways and the ligand polymorphisms differed substantially among the Colombian and Brazilian isolates while the Peruvian isolates represent an amalgam of those present in the Colombian and Brazilian field isolates. The NrTrCr invasion profile was associated with the presence of the PfRh2a pepC variant, the PfRh5 variant 1 and EBA-181 RVNKN variant. The ebl and Pfrh expression levels in a field isolate displaying the NrTrCr profile also pointed to PfRh2a, PfRh5 and EBA-181 as being possibly the major players in this invasion pathway. Notably, our studies demonstrate the uniqueness of the Peruvian P. falciparum field isolates in terms of their invasion profiles and ligand polymorphisms, and present a unique opportunity for studying the ability of P. falciparum parasites to expand their invasion repertoire after being reintroduced to human populations. The present study is directly relevant to asexual blood stage vaccine design focused on invasion pathway proteins, suggesting that regional invasion variants and global geographical variation are likely to preclude a simple one size fits all type of vaccine.

Figure 1. Plasmodium falciparum malaria endemicity in South America and geographic location of the sampling areas.

The P. falciparum malaria endemicity for 2010 in South America is stratified according to API into stable transmission (dark grey areas; PfAPI ≥0.1 per 1000 per annum), unstable transmission (medium grey areas; PfAPI<0.1 per 1,000 per annum) and no risk or malaria free (light grey and white areas; PfAPI = 0) . Circles represent populations sampled for this study: Urabá, Colombia (blue circles); Loreto, Peru (red circles); Belém and Mato Grosso, Brazil (green and yellow circles, respectively).

Figure 2. Sialic acid (SA)-dependence of invasion by P. falciparum field isolates from South America.

Invasion via SA-dependent pathway was defined as less than 50% invasion into neuraminidase-treated RBCs. Invasion via SA-independent pathway was defined as more than 50% invasion into neuraminidase-treated RBCs. Percentage of field isolates using a SA-independent (black bars) and SA-dependent (white bars) pathway in Colombia (n = 6), Peru (n = 16) and Belém, Brazil (n = 8) or Mato Grosso, Brazil (n = 14) is compared.

Figure 3. Distribution of the eight profiles of invasion in field isolates from Peru, Colombia and Brazil.

(A) The number of isolates using a specific invasion profile based on their combined sensitivity to neuraminidase (N), trypsin (T) and chymotrypsin (C). Sensitive (s) is defined as less than 50% of invasion into enzymes-treated RBC (white box) and resistant (r) is defined as higher than 50% of invasion (gray box). (B) Percentage of invasion profiles in each area of study is presented using pie charts. Brazilian isolates include those from Belém and Mato Grosso.

Figure 4. Presence and prevalence of EBL polymorphisms in field isolates from Peru, Colombia and Brazil.

(A) Variants of EBA-181 protein were defined according the amino acid substitutions at five positions. The percentage of field isolates from Colombia, Peru and Brazil (combination of samples from Belém and Mato Grosso) with each of the variants is shown. (B) Amino acid substitutions at eight positions were used to categorize the EBA-175 variants. (C) Variants of ebl-1 gene were defined according to the absence (WT, wild type) or presence of 5 thymidines insertion at position 572 (5 T’s), and the absence of the gene after nested PCR amplification (deletion).^a Number of parasites containing the variant in each area of study is shown in parenthesis. ^bAmino position according to the reference sequence is shown. EBA-181 (GenBank accession no. AY496955; 3D7), EBA-175 (GenBank accession no. FJ655429; 3D7) and EBL-1 (GenBank accession no. AF131999; Dd2). Shaded residues differ from the strain reference sequence.^cCode used in bar graphs and text. ^dThe RBC binding profile reported per each EBA-181 variant is shown . Neuraminidase (N), trypsin (T), chymotrypsin (C), sensitive (s) and resistant (r).^e Sequence alignment of nucleotides 564–584 of the ebl-1 gene is shown.

Figure 5. Presence and prevalence of PfRh1, PfRh2a and PfRh2b polymorphisms in field isolates from Peru, Colombia and Brazil.

(A) Variants of PfRh1 were defined by the number of HN and QN repeats and amino acids deletion. The percentage of field isolates from Colombia, Peru and Brazil (combination of samples from Belém and Mato Grosso) with each of the variants is shown. (B) Haplotypes in PfRh2a were defined according to amino acid deletion (15 aa and 50 aa deletion) and the peptide sequence at position 2734–2741. (C) Variants of PfRh2b were defined according to amino acid deletion (52 aa) and the peptide sequence at position 2769–2777. ^aNumber of parasites containing the variant in each area of study is shown in parenthesis. ^bAmino position according to the reference sequence is shown. PfRh1 (GenBank accession no AF411930; HB3), PfRh2a (GenBank accession no. AY138497; 7G8) and PfRh2b (GenBank accession no. AY138500; 3D7). Shaded residues differ from the strain reference sequence.^cCode used in graphs and text. ^dDel: deletion.

Figure 6. Presence and prevalence of PfRh4 and PfRh5 polymorphisms in field isolates from Peru, Colombia and Brazil.

(A) Variants of PfRh4 were defined by the number of DEVE repeat. The percentage of field isolates from Colombia, Peru and Brazil (combination of samples from Belém and Mato Grosso) with each of the variants is shown. (B) Haplotypes in PfRh5 were defined as combination of amino acids in 10 positions reported previously as polymorphic . ^aNumber of parasites containing the variant in each area of study is shown in parenthesis. ^bAmino position according to the reference sequence is shown. PfRh4 (GenBank accession no. AF432854; 3D7); PfRh5 (GenBank accession no. PFD1145c). Shaded residues differ from the strain reference sequence.^cCode used in graphs and text. ^dIns: insertion.

Figure 7. Pfrh2a, Pfrh5 and eba-181 are the most abundantly expressed ligand transcripts infield isolates utilizing the NrTrCr invasion profile.

Each bar shows the relative proportion value for each transcript as a percentage of all seven transcripts including eba-175, eba-181, Pfrh1, Pfrh2a, Pfrh2b, Pfrh4 and Pfrh5. Values are given as means ± standard errors of the means. (A) Comparison of the transcriptional levels of the ligands between parasites utilizing NrTrCr and NsTrCr invasion profiles. (B) Comparison of the transcriptional levels of the ligands between parasites utilizing NrTrCr and NrTsCr invasion profiles.