Transcribed var genes associated with placental malaria in Malawian women

Abstract

Determining the diversity of PfEMP1 sequences expressed by Plasmodium falciparum-infected erythrocytes isolated from placentas is important for attempts to develop a pregnancy-specific malaria vaccine. The DBLgamma and var2csa DBL3x domains of PfEMP1 molecules are believed to mediate placental sequestration of infected erythrocytes, so the sequences encoding these domains were amplified from the cDNAs of placental parasites by using degenerate oligonucleotides. The levels of specific var cDNAs were then determined by quantitative reverse transcription-PCR. Homologues of var2csa DBL3x were the predominant sequences amplified from the cDNAs of most placental but not most children's parasites. There was 56% identity between all placental var2csa sequences. Many different DBLgamma domains were amplified from the cDNAs of placental and children's isolates. var2csa transcripts were the most abundant var transcripts of those tested in 11 of 12 placental isolates and 1 of 6 children's isolates. Gravidity did not affect the levels of var2csa transcripts. We concluded that placental malaria is frequently associated with transcription of var2csa but that other var genes are also expressed, and parasites expressing high levels of var2csa are not restricted to pregnant women. The diversity of var2csa sequences may be important for understanding immunity and for the development of vaccines for malaria during pregnancy.

FIG. 1.

(a) Alignment of degenerate oligonucleotides D3F, D3R1, and D3R2 with four DBLγ sequences that all bind CSA as recombinant proteins (18) and with the DBL3x domain of the ItG var2csa homologue (It4var4). Asterisks under the sequences indicate that the nucleotides at those positions of the oligonucleotides had homology with both DBLγ and DBL3X sequences. W, A or T; R, A or G; M, A or C; Y, C or T; S, G or C; I, inosine. (b) Observed and expected frequencies of detection of var2csa and different DBLγ sequences in cloned PCR products amplified from 3D7 genomic DNA using the oligonucleotides D3F and D3R1. Excel BINOMDIST was used to calculate the expected binomial distribution for 40 sequenced clones with 15 DBLγ or DBL3x possible identities if each identity was equally likely (38).

FIG. 2.

Phylogenetic tree of sequences amplified from cDNAs and gDNAs of parasites isolated from placentas and from peripheral blood of children. The candidate CSA-binding PfEMP1s encoded by var2csa (35), FCR3.varCSA (6), var-CS2 (29), MAL6P14 (17), 3D7chr5var (32), 482, 498, 485, 720, and 732 (7, 23) are included, and those shown in bold are in the clusters described in the text. Bootstrap values of >50 are indicated at tree branches leading to clusters of sequences. The sequences used as endogenous DBLγ controls for Q-RT-PCR are indicated with stars. The sequences were named for the samples from which they were amplified, followed by a space and then a different number for each different sequence from a single Mplc or Mch sample or a different letter for each different sequence from an Rplc sample. The suffix “-g” following an Mplc or Mch sequence indicates that it was amplified from gDNA.

FIG. 3.

Percentage that each var gene constituted of the total var cDNA for the three or four var genes examined in each isolate by Q-RT-PCR absolute quantitation. Three var genes were quantitated for Mplc78 and for the five placental isolates with the prefix “(ne)-,” from which no endogenous control DBLγ sequence could be amplified; four var genes were quantitated for the other 13 isolates. Endogenous refers to the endogenous DBLγ sequence that was amplified from the sample being analyzed.

FIG. 4.

(a) cDNA levels in CS2 parasites of var2csa, 18S rRNA, and the two genes, PF11_0505 and the SBP gene (PFE-0065w), that were used to normalize the Q-RT-PCR data. (b) Absolute quantities of var cDNA levels in placental (Mplc) and children's (Mch) isolates were determined by Q-RT-PCR, using standard curves, and the data were then normalized using the levels of SBP and PF11_0505 cDNAs. Consequently, the data shown represent the cDNA levels of specific var genes in equivalent amounts of total var cDNA from each isolate, as estimated using two control genes that approximate the transcriptional profile of var genes. The gravidity of the patient from whom each placental isolate was obtained is indicated in parentheses after the title of the isolate.Endogenous refers to the endogenous DBLγ sequence that was amplified from the sample being analyzed. The prefix “(ne)-” indicates that no endogenous DBLγ sequence control was available for that sample.