Evolution of transcriptional control of antigenic variation and virulence in human and ape malaria parasites

Abstract

Background: The most severe form of human malaria is caused by the protozoan parasite Plasmodium falciparum. This unicellular organism is a member of a subgenus of Plasmodium called the Laverania that infects apes, with P. falciparum being the only member that infects humans. The exceptional virulence of this species to humans can be largely attributed to a family of variant surface antigens placed by the parasites onto the surface of infected red blood cells that mediate adherence to the vascular endothelium. These proteins are encoded by a large, multicopy gene family called var, with each var gene encoding a different form of the protein. By changing which var gene is expressed, parasites avoid immune recognition, a process called antigenic variation that underlies the chronic nature of malaria infections.

Results: Here we show that the common ancestor of the branch of the Laverania lineage that includes the human parasite underwent a remarkable change in the organization and structure of elements linked to the complex transcriptional regulation displayed by the var gene family. Unlike the other members of the Laverania, the clade that gave rise to P. falciparum evolved distinct subsets of var genes distinguishable by different upstream transcriptional regulatory regions that have been associated with different expression profiles and virulence properties. In addition, two uniquely conserved var genes that have been proposed to play a role in coordinating transcriptional switching similarly arose uniquely within this clade. We hypothesize that these changes originated at a time of dramatic climatic change on the African continent that is predicted to have led to significant changes in transmission dynamics, thus selecting for patterns of antigenic variation that enabled lengthier, more chronic infections.

Conclusions: These observations suggest that changes in transmission dynamics selected for significant alterations in the transcriptional regulatory mechanisms that mediate antigenic variation in the parasite lineage that includes P. falciparum. These changes likely underlie the chronic nature of these infections as well as their exceptional virulence.

Keywords: Cytoadherence; Mutually exclusive expression; Pathogenesis; Transcriptional regulation.

Fig. 1

Conservation of var intron structure in all seven Laverania species. A Schematic showing the division of var introns into three regions based on base content and strand asymmetry. B Calculation of base content of the positive strand of var introns for each species of the Laverania. Note the prominent G vs C asymmetry in regions 1 and 3 and the conservation of this asymmetry in all seven species

Fig. 2

Conservation of the unusual var gene var1csa in P. reichenowi, P. praefalciparum and P. falciparum. A A phylogenetic tree of the Laverania as described by Otto et al. [12]. B Identification of var1csa in P. falciparum (isolate SN1) and its orthologues in P. reichenowi and P. praefalciparum. The chromosomal region from each species containing the var gene with the highest sequence identity to var1csa from P. falciparum is shown. var genes are shown in blue and members of other multicopy gene families are shown in green. The genes with the highest sequence identity to var1csa in each species are aligned, and the percentage identity is shown below the gene. For P. reichenowi and P. praefalciparum, sequence identity exceeds 90%, and the genes display clear synteny (boxed in blue). For the remainder of the Laverania, the sequence identity is substantially lower and the most similar gene is not located in the syntenic position, indicating these species do not contain true orthologues of var1csa

Fig. 3

Conservation of the var gene var2csa in P. reichenowi, P. praefalciparum and P. falciparum. A Schematic showing the syntenic region of chromosome 12 in P. reichenowi, P. praefalciparum and three isolates of P. falciparum (SN01, 7G8 and 3D7). The positions of the single copy genes fikk12 and acs7 are shown and represent the border between the core genome and the highly variable subtelomeric domain. The orthologues of var2csa are aligned and boxed in blue. B Alignment of the amino acid sequence encoded by the uORF of var2csa from P. praefalciparum, P. reichenowi and three isolates of P. falciparum (SN01, 7G8 and 3D7)

Fig. 4

Maximum-likelihood phylogenetic tree of 0.5–1.5 kb upstream regulatory regions of var genes from seven Laverania species. The annotation number for each gene (from Plasmodb.org) is shown and the colour of the text signifies the species, as shown in the lower right panel. Sequences of the UpsB type are shaded in pink, UpsA in light blue, UpsC in yellow, var2csa in gray and var1csa in dark blue. Five genes with atypical upstream sequences from P. falciparum that segregate outside of the UpsA/B/C/D/E groups are marked with an asterisk. The evolutionary history was inferred using the Maximum Likelihood method and Jukes–Cantor model [63], with bootstrap values for 1000 replicates shown for various nodes. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. Evolutionary analyses were conducted in MEGA X [64]

Fig. 5

Phylogenetic trees marking key moments in the evolution of the var gene family. A The evolutionary relationships of the Laverania are shown, with each parasite species and its ape host denoted. var genes, the regulatory intronic element and the putative regulatory protein PTEF are thought to have originated in the common ancestor of the entire subgenus (shown by green asterisk). A significant shift in the structure of the var encoded protein EMP1 and the UpsC upstream regulatory type occurred in the common ancestor of the bottom five species (shown by red asterisk) while the evolution of the conserved genes var1csa and var2csa, as well as the UpsA and B types, occurred prior to the branch that includes the human parasite P. falciparum (shown by purple asterisk). B The codivergence model of parasite speciation is displayed showing the branch of the Laverania that evolved var1csa, var2csa and the UpsA/B/C organization. The phylogenetic tree shows the evolution of gorillas, humans, chimpanzees and bonobos from a common ancestor ~ 8–9 million years ago. This model presumes that parasites species diverged along with their hosts, resulting in P. praefalciparum, P. reichenowi and P. lomamiensis infecting gorillas, chimpanzees and bonobos, respectively. Humans are hypothesized to have lost their original parasites, then become reinfected through a recent gorilla-to-human transmission event, resulting in P. falciparum. These ape species (and their parasites) initially diverged during the late Miocene, a period of major climatic change in Africa marked by aridification and a regional shift from rainforests to grasslands and savannah