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. 2016 Apr 4:7:11187.
doi: 10.1038/ncomms11187.

Ancient human sialic acid variant restricts an emerging zoonotic malaria parasite

Selasi Dankwa  1 , Caeul Lim  1 , Amy K Bei  1 , Rays H Y Jiang  1 , James R Abshire  2 , Saurabh D Patel  1   3 , Jonathan M Goldberg  1 , Yovany Moreno  1 , Maya Kono  1 , Jacquin C Niles  2 , Manoj T Duraisingh  1
Affiliations

Ancient human sialic acid variant restricts an emerging zoonotic malaria parasite

Selasi Dankwa et al. Nat Commun. .

Abstract

Plasmodium knowlesi is a zoonotic parasite transmitted from macaques causing malaria in humans in Southeast Asia. Plasmodium parasites bind to red blood cell (RBC) surface receptors, many of which are sialylated. While macaques synthesize the sialic acid variant N-glycolylneuraminic acid (Neu5Gc), humans cannot because of a mutation in the enzyme CMAH that converts N-acetylneuraminic acid (Neu5Ac) to Neu5Gc. Here we reconstitute CMAH in human RBCs for the reintroduction of Neu5Gc, which results in enhancement of P. knowlesi invasion. We show that two P. knowlesi invasion ligands, PkDBPβ and PkDBPγ, bind specifically to Neu5Gc-containing receptors. A human-adapted P. knowlesi line invades human RBCs independently of Neu5Gc, with duplication of the sialic acid-independent invasion ligand, PkDBPα and loss of PkDBPγ. Our results suggest that absence of Neu5Gc on human RBCs limits P. knowlesi invasion, but that parasites may evolve to invade human RBCs through the use of sialic acid-independent pathways.

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Figures

Figure 1
Figure 1. Expression of chimpanzee CMAH in human red blood cells (RBCs) introduces the non-human sialic acid variant Neu5Gc on the cell surface.
(a) The phylogenetic tree for a range of primates indicates loss of CMAH (highlighted in red) and timing of this event in ancestral hominins and New World monkeys (marmoset and Saimiri monkey). Myr ago, million years ago. (b) Domain structures of pLVX-Puro and PtCMAH.pLVX-Puro. Ψ, packaging signal; CMV IE p, cytomegalovirus immediate early promoter; cPPT, central polypurine tract; PGK p, phosphoglycerate kinase promoter; PuroR, puromycin resistance gene; RRE, Rev-response element; WPRE, woodchuck hepatitis virus post-transcriptional regulatory element. (c) Normal morphology of day 21 pLVX and PtCMAH cRBCs. Scale bar, 10 μm. (d) Neu5Gc expression on RBCs as measured by flow cytometry using an α-Neu5Gc antibody. Shown are representative plots from at least six independent experiments. Normalized to mode—normalization to the modal fluorescence value. (e) Neu5Gc expression on RBCs as measured by western blot (left panel). The coomassie-stained protein gel (right panel) shows total protein from RBC samples analysed by western blot. Representative images are shown from two independent experiments. (f) Relative amounts of Neu5Ac and Neu5Gc on RBCs by HPLC analyses. The average of three independent experiments is shown. Error bars represent the s.e.m.
Figure 2
Figure 2. Introduction of Neu5Gc on human RBCs increases P. knowlesi invasion but does not affect P. falciparum invasion.
(a) P. knowlesi H invasion of PtCMAH cRBCs is significantly enhanced, compared with pLVX cRBCs. (b,c) Sialic acid-dependent and sialic acid-independent P. falciparum strains Dd2 (b) and 3D7 (c), respectively, invade PtCMAH cRBCs and pLVX cRBCs similarly. Human and macaque RBCs are included as controls for each strain. The parasitized erythrocyte multiplication rate for P. knowlesi H is the average of four independent experiments and for P. falciparum Dd2 and 3D7, three independent experiments. Error bars indicate the s.e.m. The Student's t-test (two-tailed) was used to assess significant differences in invasion. *P=0.012. NS, not significant.
Figure 3
Figure 3. P. knowlesi invasion ligands PkDBPβ and PkDBPγ bind to Neu5Gc-sialylated receptors.
(a) Left panel—PkDBPα-COMP binds to DARC on untreated (U), neuraminidase-treated (N) and trypsin-treated (T) macaque RBCs, but not chymotrypsin-treated (C) macaque RBCs as seen in a protein overlay. Right panel—α-DARC western blot confirms that the PkDBPα receptor on macaque RBCs is DARC (highlighted by the bracket). (b) Binding of PkDBPβ-COMP and PkDBPγ-COMP to untreated and protease-treated macaque RBCs, but not to neuraminidase-treated macaque RBCs. GST-COMP does not bind to macaque RBCs. Data are representative of at least four independent experiments. (c) PkDBPβ-COMP (red) and PkDBPγ-COMP (blue) bind to untreated (solid trace) PtCMAH cRBCs, as to macaque RBCs, but not to pLVX cRBCs, like human RBCs as determined by a flow cytometry-based assay. Binding to neuraminidase-treated (dashed trace) PtCMAH cRBCs and macaque RBCs is markedly decreased. COMP protein (black) does not bind to any cell type. The assay was performed twice, in triplicate; representative traces are shown.
Figure 4
Figure 4. Neu5Gc-independent invasion of human-adapted P. knowlesi YH1 strain.
(a) A human-adapted laboratory strain, Pk YH1, invades Neu5Gc-positive and Neu5Gc-negative RBCs similarly. Shown is the average PEMR of three biological replicates performed in triplicate. Error bars represent the s.e.m. NS, not significant (Student's t-test (two-tailed)). (b) Read coverage normalized to the average genome-wide coverage for regions containing the DBP loci in Pk YH1 and the parental Pk H strain. Pk YH1 has a PkDBPα duplication (left panel) and a PkDBPγ deletion (bottom panel). PkDBPβ in Pk YH1 is unchanged (right panel). The intron–exon structure of each gene is shown above the corresponding chromosomal region. (c) The IC50 of MGSA against Pk YH1 invasion of human RBCs is increased >10-fold compared with Pk H. Shown is the average invasion efficiency of three (Pk H) or four (Pk YH1) biological replicates. Error bars indicate the s.e.m.
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