Abstract: Plasmodium species are frequently host-specific, but little is currently known about the molecular factors restricting host switching. This is particularly relevant for P. falciparum, the only known human-infective species of the Laverania sub-genus, all other members of which infect African apes. Here we show that all tested P. falciparum isolates contain an inactivating mutation in an erythrocyte invasion associated gene, PfEBA165, the homologues of which are intact in all ape-infective Laverania species. Recombinant EBA165 proteins only bind ape, not human, erythrocytes, and this specificity is due to differences in erythrocyte surface sialic acids. Correction of PfEBA165 inactivating mutations by genome editing yields viable parasites, but is associated with down regulation of both PfEBA165 and an adjacent invasion ligand, which suggests that PfEBA165 expression is incompatible with parasite growth in human erythrocytes. Pseudogenization of PfEBA165 may represent a key step in the emergence and evolution of P. falciparum

Dankwa, Selasi

Duraisingh, Manoj T.

Hahn, Beatrice H.

Kemp, Alison

Liu, Weimin

Marsden, Sarah

Proto, William R.

Rayner, Julian C.

Sharp, Paul M.

Siegel, Sasha V.

Unwin, Steve

Wright, Gavin J.

Zenonos, Zenon A.

Apollo (Cambridge)

Adaptation of Plasmodium falciparum to humans involved the loss of an ape-specific erythrocyte invasion ligand

Supplementary Information Adaptation of Plasmodium falciparum to humans involved the loss of an ape-specific erythrocyte invasion ligand Proto et al      Supplementary Figure 1. PfEBA165 full alignment. Exemplar EBA165 amino acid sequences (~800 bp amplified fragment) from ape-infective Laverania species (G1, Plasmodium praefalciparum; G2, P. adleri; G3, P. blacklocki; C1, P. reichenowi; C2, P gaboni; C3, P. billcollinsi; B1, P. lomamiensis) as recently described 3. The 3D7 P. falciparum sequence shown is that which would be translated if both frameshifts were corrected, and represents the relevant fragment of the full-length corrected PfEBA165 protein ectodomain that was expressed recombinantly.   Alignment: newfileSeaview [blocks=80 fontsize=10 A4] on Thu Sep 27 12:15:57 2018    1Pf  ALCKLENYIKNEYDRENSFYILNNKVEKEYEKGT-NNNINIDKLANGSLEMLDK-KEALLEDKKNVDNKDIKEQLPENNKG1  ALCKLENYIKNEYDRENSFYILNNKVEKEYEKGT-NNNINIDKLANGSLEMLEVKKEALLEDKKNVDNKDIKEQLPEHNKG2  TLCKLENYLKNEYEKENSFNIINNKVEKEYDSGT-NNNVNIDSSSNGKLELLEVKEDALLND------KNIQEQEAENNIC1  ALCKLENYIKNEYDKENSVYIINNKVEKDYGSGMNNNNINIDKLPNGSLEILKVKKEALLGDKKNIDNKDIKEQLPENNKC2  TLCKLENYLKNEYEKENSFNIINNKVVKEYDSGT-NNNVNIDRSSNGRLELLEVKEEALLND------KNIEEEEAENNIC3  TLCKLENNIKNKYDKENSFYIINNKVEKAYEGGT-NNNINIDKLSNGSLEMLEVKEETLFDDKKNIHNKDIKEQLTENNIB1  ALCKLENYIKNEYES-----------------GM-NNNINIDKLPNGSLEILKVKKEALLGDKKNIDNKDIKEQLPENNK   81Pf  NFEEKILHEDICKLNKENIEKEGLYKPNTFKSIGNDLLYKKGKLNFLYEYGMELLHINKVPMNRMNNSRGLGDSNMSFLDG1  NFEEKILYEDICKLNKENIEKEGLYKPNTFKSIGNDLLYKKGKLNFLYEYGMELLHINKVPMNRMNNSRGLGDSNMSFLDG2  NFEEKILHKNVYELNKESIEKEGLYKPNTIKNIEDESLYKKGKLNFLYEYGMELLNINKVRMNKMNNSRGLGDSNTSFLDC1  NFEEKILHKDICKLNKENIEKEGLYKPNTFKNIENDLLYKKGKLNFLYDYGMELLHINKAPMNRMNNSRGLGDGDMSFLDC2  NFEEKILYKNVCELNKENIEKEGLYKPNTIKNIENDSLYKKGMLNFLYEYGMELLNINKVPMNKMNNSRVLGDSKMSFLDC3  NYEEKNLLKDICILNKEKIEKEGLYKPSTFKYIENDSLYKKGKLNFLYVYGMELLHINKTSMNRMNNFRVLGDSNMSYLDB1  NFEEKILHKDICKLNKENIENEGLYKPNTFKNIENDLLYKKGKLNFLYEYGMELLHINKARMNRMNNSRGLGDSNMSFLD  161Pf  ISNNNMNSLNSM---NNLNNSNNFDSSNSLSFVDMKSIHRCIKKRAKGERDWACNDKNTKEPNICVSDRRVQLCTGNLIEG1  ISNNNMNSLNNLNNLNNLNNSNNFDSSNSLSFVDMKSIHRCIKKRAKGERDWACNDKNTKEPNICVSDRRVQLCTGNLIEG2  ISNNNNKN-----------KNNNMNNLNSLSFVDMKSVHRCITKRKKGEKDWACNDKNTKEPNICVSDRRVQLCTGNLVDC1  ISNNNMNISNSL------------DSLNSLSFVDMKSIHRCIKKRAKGERDWVCNNKNTKEPNICVSDRKVQLCTGNLIEC2  ISNNNNKN-----------KNNNMNNLNSLSFVDMKSIHRCITKRKKGEKDWACNDKNTKEPNICVSDRRVQLCTGNLVDC3  ISNNTNN--NNMNSLNSSNNLNNMNSFNSLSFVDMKFLHRCIKKRNKGERDWKCNDINTKEPNICVSDRRVQLCTGNLIDB1  ISNNNMNILNSVDN---------LDSLNSLSFVDMKSIHRCIKKRAKGERDWVCNNKNTKEPNVCVSDRKVQLCTGNLIE  241Pf  LPINDSTKEKFKEKLILAAQKEGSLLFEKFGKKYNEEFCLNLKWSYGDYGDIIKG1  LPINDSTKDKFKEKLILAAQKEGSLLFEKFGKKYNEEFCLNLKWSYGDYGDIIKG2  LSIDESTKEKFKEKLIIAAKREGTLLFEKFGKEYTAEFCLNLKWSYSDYGDIIKC1  LPISDSTKEKFKEKLILAAQKEGSLLFEKFGKKYNEEFCLNLKWSYGDYGDIIKC2  LSINDATKEKFEGKLIIAAKREGTLLFEKFGKKYTAEFCLNLKWSYSDYGDIIKC3  IPTNESTKEKFKEKLILAAKREGSLLFEKFGKIYNAEFCLNLKWSYSDYGDIIKB1  LPISDSTKEKFKEKLILAAEKEGNLLFEKFGKKYNEEFCLNLKWSYGDYGDIIK Supplementary Figure 2. PfEBA165, PrEBA165 and PfEBA175 bind primarily sialic acid containing glycans Purified mono-biotinylated glycans (GlycoTech) were immobilised on streptavidin-coated 96 well plates and probed with normalised recombinant pentameric β-lactamase-tagged PfEBA165, PrEBA165 and PfEBA175 (black, grey, and white bars, respectively). Binding was detected by incubation with nitrocefin, a substrate for β-lactamase. As expected from the known binding specificity of PfEBA175, most binding was detected with glycans containing sialic acids. Data points represent mean values of three technical replicates, conducted on the same batches of recombinant proteins and glycans.    Supplementary Figure 3. CMAH expression has no impact on surface levels of major erythrocyte receptors. Surface expression of Basigin (BSG), Glycophorin C (GPC), Glycophorin A (GPA), CD71, Duffy Antigen Receptor for Chemokines (DARC) was measured by incubating cultured red blood cells (cRBCs) or human red blood cells (RBCs) with either commercial monoclonal antibodies followed by fluorescently tagged secondary antibodies (BSG and Neu5Gc), or primary antibodies directly conjugated to fluorophores (DARC, CD71, GPA, GPC), with binding detected by flow cytometry (blue traces). Secondary antibodies alone were used as a negative control for binding (red traces). There was no detectable difference in the surface expression of known P. falciparum invasion receptors (BSG, GPA, GPC) between cRBCs produced from hSCs transfected with control (pLVX) or CMAH expressing vectors. cRBCs expressed higher levels of CD71 and lower levels of DARC compared to human RBCs, as has been previously shown with the cRBC expression system, thought to reflect the fact that cRBCs represent more immature RBCs1,2. Expression of CMAH resulted in Neu5Gc expression on the cRBC surface. NeuGc is present in primate erythrocytes such as ape and macaque (shown), but not on human erythrocytes or control cRBCs.    Supplementary Figure 4. Genome editing of PfEBA165 to correct frameshift mutations has no impact on erythrocyte invasion profile or in vitro growth rate. A) Relative growth of P. falciparum 3D7 (parental), YB4 (cloned transfectant with 1 frameshift corrected) and C10 and D3 (cloned transfectant lines with both frameshifts corrected) lines during continuous culture in human erythrocytes. Lines were diluted with fresh red blood cells every two days, parasitaemia counted using flow cytometry, and cumulative parasitaemia calculated adjusting for the dilution factors. There was no difference in growth rates between the lines. Data points represents mean values of three biological replicates. Error bars represent standard deviation. B) Invasion of P. falciparum 3D7 (parental), and C10 and D3 (cloned transfectant lines with both frameshifts corrected) into human erythrocytes that were either untreated (u), or treated with neuraminidase (NM) to remove surface sialic acids. Erythrocytes were labelled with fluorescent dyes, and new invasion events were identified using a two-colour flow cytometry assay3. Two separate assays were conducted (expt 1 and expt 2). Invasion rates are expressed relative to the invasion into untreated human erythrocytes of each strain in separate experiments. Bars represent mean values of four technical replicates for expt 1 and three technical replicates for expt 2, with individual data points overlaid as black circles. Error bars represent standard deviation.  C) Invasion of P. falciparum 3D7 (parental), and C10 and D3 (cloned transfectant lines with both frameshifts corrected) into chimpanzee erythrocytes that were either untreated (u), or treated with neuraminidase (NM) to remove surface sialic acids. Erythrocytes were labelled with fluorescent dyes, and new invasion events were identified using a two-colour flow cytometry assay3. Invasion rates are expressed relative to the invasion into untreated chimpanzee erythrocytes of each strain. Bars represent mean values of four technical replicates from one assay, with individual data points overlaid as black circles. Error bars represent standard deviation. D) P. falciparum 3D7 (parental), and C10 and D3 (cloned transfectant lines with both frameshifts corrected) lines were incubated with enzyme treated erythrocytes for 24 hours, and invasion measured using two-colour flow cytometry to count invasion only into treated erythrocytes. Data points represent mean values of three technical replicates from a single assay. Error bars represent standard deviation. Invasion rates are expressed relative to invasion into untreated erythrocytes for each parasite line.         Supplementary Figure 5. Flow cytometry gating strategy for erythrocyte binding assays.  Gates were determined using control samples with cells only. Cells were selected using gates P1 (side scatter-area (SSC-A) vs forward scatter-area (FSC-A)), P2 (forward scatter-width (FSC-W) and forward scatter-area (FSC-A)), and P3 (forward scatter-area (FSC-A) and phycoerthrin-area (PE-A)).   Supplementary Table 1. Significantly down-regulated genes in PfEBA165 corrected lines. Significantly down-regulated gene clusters in PfEBA165 corrected lines (double-edited clones C10 and D3). Genes that are significantly down-regulated (p-value ≤ 0.05) in both C10 and D3 relative to the parent line 3D7 in the order are listed in which they appear along the two down-regulated clusters of genes located on chromosomes 4 and 11, respectively.    Table 1. Significantly differentially expressed gene regions in double-edited clones C10 and D3 GeneID Gene descriptionLog2FCC10p-valueC10Log2FCD3p-valueD3Pf3D7_0424200 Reticulocyte binding protein homologue (Rh4)-1.03 3.32E-42 NA NAPf3D7_0424300 Erythrocyte binding antigen 165 (EBA165)-2.20 8.33E-137 -2.18 3.41E-78Pf3D7_0424400 Surface-associated interspersed protein 4.2 (SURFIN 4.2)-1.28 0.028 -1.78 0.003Pf3D7_0424500 Serine/threonine protein kinase,FIKK family-1.25 3.33E-14 -1.23 3.48E-10Pf3D7_0424600 Plasmodium exported protein (PHISTb, unknown function)-0.89 2.87E-26 -0.69 0.001Pf3D7_0424700 Serine/threonine protein kinase, FIKK family-0.65 7.81E-05 -1.44 1.16E-10Pf3D7_0424800 Plasmodium exported protein (PHISTa, unknown function)-2.27 0.0007 -1.84 0.003Pf3D7_0424900 Plasmodium exported protein (PHISTb, unknown function)-1.18 8.08E-08 -1.64 1.08E-08Pf3D7_1147000 Sporozoite and liver stage asparagine-rich protein (SLARP)-6.29 9.31E-234 -6.62 1.13E-159Pf3D7_1147100 Dynein light chain, putative -3.69 0.0006 -3.44 0.0004Pf3D7_1147200 Tubulin tyrosine ligase,putative -3.74 2.43E-110 -3.16 2.87E-54Supplementary Table 2. Oligonucleotides used in this study    Oligo Description Gene name Gene ID 5' to 3' sequence OL176 gRNA 5' frameshift.  Anneal to OL177. PfEAB165 PF3D7_0424300 ATTGATAAGTTGGCAAATGGGAGTT OL177 gRNA 5' frameshift.  Anneal to OL176 PfEAB165 PF3D7_0424300 AAACAACTCCCATTTGCCAACTTAT OL186 Phosphorothoiate modified oligo. Product for transfection, targets 5' frameshift. PfEAB165 PF3D7_0424300 C*A*AGAAAGAAAGAAAAACAAGC OL187 Phosphorothoiate modified oligo. Product for transfection, targets 5' frameshift. PfEBA165 PF3D7_0424300 G*T*ACTCCCTTCTTTTTGTGC OL231 gRNA 3' frameshift.  Anneal to OL232. PfEAB165 PF3D7_0424300 ATTGAACATAAAGAGAAATTTCCAA OL232 gRNA 3' frameshift.  Anneal to OL231. PfEAB165 PF3D7_0424300 AAACTTGGAAATTTCTCTTTATGTT OL257 Phosphorothoiate modified oligo. Product for transfection, targets 3' frameshift. PfEAB165 PF3D7_0424300 C*A*AGAGAAATTGATTTTGGCAGC OL258 Phosphorothoiate modified oligo. Product for transfection, targets 3' frameshift. PfEBA165 PF3D7_0424300 G*T*TCAAGATACATATTCTCATGC OL293 Amplify PCR product for sequencing PfEAB165 PF3D7_0424300 TTAACCCTGATTTTGATGC OL271 Amplify PCR product for sequencing PfEAB165 PF3D7_0424300 TCTCTCCACATATCTTTACC OL265 qRT-PCR PfAMA1 PF3D7_1133400 CAGCTGCTGTCGCTGTATTA OL266 qRT-PCR PfAMA1 PF3D7_1133400 CCATAATCTTGTGGTTCATCCATTT OL307 qRT-PCR PfEBA165 PF3D7_0424300 AGGTGGCACACAAAGTAGTC OL308 qRT-PCR PfEBA165 PF3D7_0424300 CAGTGCTTGGATCTTCTCTACC OL315 qRT-PCR Pfcyp87 PF3D7_0510200 AAACGGGAGATCCTTCAGGT OL316 qRT-PCR Pfcyp87 PF3D7_0510200 AAGGACATGGGACAGTGGTT      References:  1. Dankwa, S., Lim, C., Bei, A. K., Jiang, R. H. Y., Abshire, J. R. et al. Ancient human sialic acid variant restricts an emerging zoonotic malaria parasite. Nat. Commun. 7, 11187 (2016). 2. Giarratana, M. C., Rouard, H., Dumont, A., Kiger, L., Safeukui, I. et al. Proof of principle for transfusion of in vitro–generated red blood cells. Blood 118, 5071-5079 (2011). 3. Theron, M., Hesketh, R. L., Subramanian, S. & Rayner, J. C. An adaptable two-color flow cytometric assay to quantitate the invasion of erythrocytes by Plasmodium falciparum parasites. Cytometry A 77, 1067-1074, doi:10.1002/cyto.a.20972 (2010).  

English

Plasmodium species are frequently host-specific, but little is currently known about the molecular factors restricting host switching. This is particularly relevant for P. falciparum, the only known human-infective species of the Laverania sub-genus, all other members of which infect African apes. Here we show that all tested P. falciparum isolates contain an inactivating mutation in an erythrocyte invasion associated gene, PfEBA165, the homologues of which are intact in all ape-infective Laverania species. Recombinant EBA165 proteins only bind ape, not human, erythrocytes, and this specificity is due to differences in erythrocyte surface sialic acids. Correction of PfEBA165 inactivating mutations by genome editing yields viable parasites, but is associated with down regulation of both PfEBA165 and an adjacent invasion ligand, which suggests that PfEBA165 expression is incompatible with parasite growth in human erythrocytes. Pseudogenization of PfEBA165 may represent a key step in the emergence and evolution of P. falciparum

Proto, William R

Siegel, Sasha V

Zenonos, Zenon A

Sharp, Paul M

Wright, Gavin J

Hahn, Beatrice H

Duraisingh, Manoj T

Rayner, Julian C

Adaptation of Plasmodium falciparum to humans involved the loss of an ape-specific erythrocyte invasion ligand.

Edinburgh Research Explorer

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https://www.pure.ed.ac.uk/ws/files/116678890/s41467_019_12294_3.pdf

Adaptation of Plasmodium falciparum to humans involved the loss of an ape-specific erythrocyte invasion ligand

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