Investigation of CMAH gene loss in mammalian evolution

We examined hominid and mammalian genomes with genome alignments and gene loss analysis. The mammalian gene loss events were analysed with the program CAFE (Computational Analysis of gene Family Evolution51), a statistical tool for computing the gene family size evolution that is implemented in Ensembl 72. The gene loss event in hominins was further verified with independent genomic analysis; the sequence reads of the Neanderthal52 and Denisovan genomes24 were aligned to chimpanzee genome panTro3 at the CMAH gene locus with the University of California, Santa Cruz (UCSC) genome browser53.

The ferret54 and New World monkey25 CMAH gene loss events are published. Dog CMAH does not appear to be functional in certain breeds of dogs55,56.

Ex vivo culture of RBCs

Bone-marrow-derived CD34+ haematopoietic stem cells (2–3 × 105; Lonza) were cultured in media with 5% solvent/detergent virus-inactivated human plasma (Octaplas, Octapharma) as described previously57 with the following modifications. On day 6 or 7 of culture, cells were transduced with lentivirus harbouring either the chimpanzee (Pan troglodytes) CMAH complementary DNA (cDNA) sequence in pLVX-Puro (Clontech) or the empty vector, pLVX-Puro. The CMAH cDNA sequence with a C-terminal tobacco etch virus (TEV) cleavage site and FLAG-c-myc tags was codon-optimized for human expression and synthesized (GeneArt). CMAH cDNA was amplified using primers listed in Supplementary Table 3 and cloned into pLVX-Puro. Transduction with lentiviral vectors and subsequent selection on 2 μg ml−1 puromycin dihydrochloride (Sigma-Aldrich) were done as described23, but puromycin was maintained until day 12 or 13 when cells were co-cultured on a murine MS-5 stromal cell layer at a cell density of 3–6 × 105 cells per ml until day 18 as described58. On day 18, cells were replated on a fresh MS-5 stromal cell layer and then collected on day 20 or 21 at which time 70–90% of cells were enucleated. Cells were stored at 4°C in incomplete RPMI; (consisting of RPMI-1640 (Sigma-Aldrich) supplemented with 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and 50 mg l−1 hypoxanthine), until use in downstream experiments.

Neu5Gc or RBC receptor expression by flow cytometry

RBCs and cRBCs were washed three times in PBS containing 0.5% Neu5Gc-free blocking agent (Siamab, formerly Sialix) and pelleted at 500 g for 4 min in a 96-well plate at 5 × 105 cells per well. Cells were resuspended in either 50 μl of blocking buffer or 50 μl of the appropriate antibody solution. The following antibodies were used at the indicated dilutions: anti-Neu5Gc (1:5,000, Siamab), phycoerythrin(PE)-conjugated anti-DARC (1:10, Miltenyi Biotec), anti-CD71-PE (1:10, Miltenyi Biotec), fluorescein isothiocyanate-conjugated anti-glycophorin A (GPA); 1:50, Clone 2B7, STEMCELL Technologies) and anti-glycophorin C (GPC)-fluorescein isothiocyanate (1:500, BRIC 10, Santa Cruz). Cells were incubated for 1 h at room temperature and washed three times in blocking buffer. Unstained cells and cells stained with DARC, CD71, GPA and GPC antibodies were resuspended in 100 μl PBS for analysis on the MACSQuant flow cytometer (Miltenyi Biotec). Neu5Gc-stained cells were incubated in anti-chicken IgY-Alexa Fluor 488 secondary antibody (Life Technologies) at 1:1,000 for 30 min at room temperature. Control samples were similarly incubated in anti-mouse IgG2a-PE (1:10, Miltenyi Biotec), anti-chicken IgY-Alexa Fluor 488 antibody or anti-mouse IgG-Alexa Fluor 488 (1:1,000, Life Technologies). Cells were washed twice and subjected to flow cytometric analysis. The data were analysed in FlowJo 4 version 10.0.7 (Tree Star).

Measurement of sialic acid by HPLC

For measurement of cell surface sialic acid, RBC and cRBC ghosts were prepared for sialic acid release as described59, with some modifications. In brief, 1 × 107 cells were washed once in incomplete RPMI and twice in PBS pH 7.4. Cells were stored at −80 °C until RBC ghost preparation, where thawed RBCs were resuspended in cold 5 mM sodium phosphate pH 8.0 containing protease inhibitor cocktail. RBC ghosts were pelleted at 21,130 g for 10 min, washed three times in PBS containing protease inhibitor cocktail and stored at −80 °C until sialic acid preparation. Sialic acid release from RBC ghosts was achieved by mild acid hydrolysis as described60, with some modifications. In brief, RBC ghosts were resuspended in 75 μl 2 M acetic acid and heated at 80 °C for 3 h. For formation of fluorescent derivatives, 75 μl of derivatization solution was added to sialic acid samples and heated at 50 °C in the dark for 2.5-3 h. The derivatization solution was made up of 7 mM 1,2-diamino-4,5-methylenedioxybenzene dihydrochloride, acetic acid (1.7 M for experimental samples or 3.7 M for sialic acid standards), 0.75 M β-mercaptoethanol and 18 mM sodium hydrosulfite. Samples were run on an HPLC with a C18 column at a flow rate of 0.9 ml min−1, using a mobile phase of acetonitrile–methanol–water (9:7:84 v/v). Sialic acid elution was monitored by absorbance at 373 nm. Data were acquired on Chemstation Rev A. 10.01 (Agilent Technologies). Eluted sialic acids were compared with a sialic acid reference panel, and Neu5Ac and Neu5Gc solutions of known amounts for quantitation of sialic acids.

For measurement of sialic acid in invasion assays, neuraminidase treatment of RBCs was performed in 40 μl total volume. Supernatants were collected and sialic acid was quantified as described previously61. In brief, cell supernatants were treated with 20 μl of a freshly prepared solution of 0.2 M sodium periodate in 48% phosphoric acid for 20 min at room temperature. To terminate the reaction, 100 μl of freshly prepared 10% sodium arsenite in 0.1 N sulphuric acid was added slowly, then vortexed until clear and incubated 5 min at room temperature. Finally, 600 μl of thiobarbituric acid (6 mg ml−1) was added, and the reaction was incubated at 100 °C for 15 min, then chilled on ice. Samples were analysed on the HPLC as described above, but elution was performed using a running buffer consisting of 115 mM sodium perchlorate, 30% methanol and 1% phosphoric acid, and absorbance was monitored at 549 nm.

Plasmodium parasite cultures

P. falciparum Dd2attB/BSD-GFP (BSD: blasticidin S-deaminase) and 3D7attB/BSD-GFP62 used in cRBC/RBC invasion assays were kind gifts from David Fidock (Columbia University). Pk H SSUDattB/BSD-GFP (SSUD: small subunit of the D-type ribosomal RNA gene) was generated in our lab as follows. The targeting vector, pDEF-DSSU-hDHFR63, was integrated into Pk H (provided by A.W. Thomas; Biomedical Primate Research Center, Rijswijk) to form Pk H SSUD. The P. knowlesi d-ssu gene was then amplified using Pk H SSUD genomic DNA and primers listed in Supplementary Table 3, and then cloned into pCG6-attB62. The resulting vector, pSSUD-CG6-attB, was integrated into Pk H SSUD to form Pk H SSUDattB. In the final step, pBSD-GFP-INT-attP62 was integrated into Pk H SSUDattB. Pk YH1 was generated in our laboratory from the parental Pk H strain by growth for 3 months in human O+ RBCs supplemented with 16% reticulocytes purified from haemochromatosis patient blood21. The percentage of reticulocytes was decreased to 2–4% for an additional 2 months, after which parasites proliferated stably in the absence of reticulocyte-enriched blood. P. falciparum Dd2 and W2mef strains used in invasion assays with neuraminidase-treated RBCs were obtained from the Malaria Research and Reference Reagent Resource.

P. falciparum cultures and the human-adapted Pk YH1 line were maintained in human RBCs (Research Blood Components), while P. knowlesi H SSUDattB/BSD-GFP was grown in rhesus macaque RBCs (New England Primate Research Center). Parasites were grown at 2% haematocrit in complete RPMI (RPMI supplemented with 25 mM HEPES, 50 mg l−1 hypoxanthine, 2.57 mM sodium bicarbonate and 0.5% AlbuMAX II (Life Technologies)).

Enzyme treatments

For RBC binding assays, enzyme treatments were performed as described42. In brief, RBCs or cRBCs were washed three times in incomplete RPMI and treated with 1 mg ml−1 trypsin (Sigma-Aldrich) and/or 1 mg ml−1 chymotrypsin (Worthington) and/or 66.7 mU ml−1 Vibrio cholerae neuraminidase (Sigma-Aldrich) at 37 °C with gentle mixing for 1 h. RBCs were then washed twice in incomplete RPMI and once in PBS pH 7.4.

For neuraminidase treatment for invasion assays, human RBCs were washed in wash media (RPMI supplemented with 25 mM HEPES-KOH, pH 7.4) and treated with decreasing amounts of neuraminidase from Clostridium perfringens (100, 50, 25, 12.5, 6.25 and 3.125 U; New England Biolabs) at 37 °C with gentle mixing for 30 min. The neuraminidase-treated RBCs were then washed extensively in complete RPMI before use. The neuraminidase-treated RBCs were subjected to a second neuraminidase treatment to remove and quantify the remaining sialic acid. This second treatment was performed at 250 U neuraminidase at 50% haematocrit for 30 min at 37 °C in PBS pH 6.0.

Invasion assays

For invasion assays using RBCs and cRBCs, cells were washed in complete RPMI and counted on a haemocytometer. Cells (1.5 × 106) were added to respective wells of a half-area 96-well plate at 0.5% haematocrit in triplicate. Synchronised schizont-stage parasites were purified by magnetic separation on a MACS cell separator (Miltenyi Biotec) and added to RBCs at ∼2% parasitaemia in a final volume of 30 μl. Cells (2.5 × 105; 5 μl) were taken from each well for cytospin preparations to assess initial parasitaemia by light microscopy. Assay plates were kept in a modulator incubator chamber gassed with 1% O 2 , 5% CO 2 and balance of N 2 , and reinvasion and growth were monitored over time by thin smear of mock wells. At trophozoite/schizont stage, final parasitaemia was estimated by light microscopy from counts of at least 1,000 cells on May-Grünwald- and Giemsa-stained cytospins. The parasitized erythrocyte multiplication rate (final parasitaemia (after single round of invasion)/initial parasitaemia) was determined for each strain in each cell type. The Student’s unpaired t-test (two-tailed) was used to assess significant differences in invasion.

For invasion assays with neuraminidase-treated RBCs, parasite invasion into neuraminidase-treated RBCs was measured by SYBR Green I (Life Technologies) staining and flow cytometry as described previously64, with several modifications. Synchronised schizont-stage parasites at ∼10% parasitaemia that had been treated with 250 U C. perfringens neuraminidase at ring stage to reduce reinvasion, were washed, and then mixed with untreated and neuraminidase-treated target cells. Invasion assays were seeded in triplicate 200 μl samples at ∼1% parasitaemia at 0.5% haematocrit in a 96-well plate. To quantify inoculum parasitaemia, a subset of samples was fixed immediately with formaldehyde and stored at 4 °C. The remaining samples were incubated for 48 h, then fixed for 1 h. Inoculum and post-invasion samples were then stained with SYBR Green I and analysed by flow cytometry. Expansion rates were expressed as parasitized erythrocyte multiplication rate.

For MGSA invasion inhibition assays, human RBCs were plated in a 96-well plate at 4% haematocrit in 2 × complete RPMI (complete RPMI with twice the amount of AlbuMAX II and sodium bicarbonate). MGSA (PeproTech) of varying concentrations made up in incomplete RPMI were added to respective wells. Schizont-stage parasites purified by magnetic separation, as described above, were added to each well at ∼1% parasitaemia. After allowing for a single round of invasion, parasitaemia was measured the following day by either SYBR green I or Vybrant DyeCycle Violet (Life Technologies) staining and flow cytometry. The parasitaemia of MGSA-treated samples was normalized to a mock-treated control for each strain. The data were analysed in GraphPad Prism 5.

Protein expression plasmids

Region II of PkDBPα (gene ID: PkH_062300) and Region II of PkDBPβ (gene ID: PkH_000490) were amplified from P. knowlesi H genomic DNA, while Region II of PkDBPγ (gene ID: PkH_134580) was amplified from a P. knowlesi fosmid obtained from a P. knowlesi fosmid library65, using primers listed in Supplementary Table 3. PCR products were cloned into the mammalian expression vector, pSDP32, a version of pTT66 with a modified multiple cloning site and with the rat COMP domain downstream of the multiple cloning site. For the GST-COMP-expressing plasmid, GST was amplified from pET-42a(+) (Supplementary Table 3) and cloned into pSDP32. For the COMP-negative control plasmid, NheI- and NotI-digested pSDP32 was modified with T4 DNA polymerase (New England Biolabs) to generate blunt ends and then ligated. Plasmid constructs were verified by sequencing.

HEK293-6E culture and protein expression and purification

HEK293-6E cells, a kind gift from Yves Durocher (National Research Council, Canada), were maintained in serum-free Freestyle 293 expression media (Life Technologies) supplemented with 0.1% Pluronic F-68 (Life Technologies) and 25 μg ml−1 Geneticin (Life Technologies). Cells were grown in suspension in Erlenmeyer flasks at 110 r.p.m., at 37 °C in 8% CO 2 . Recombinant protein expression by transient transfection was achieved as described66 with some modifications. In brief, cells were transfected at a cell density of 1 × 106 cells per ml using 1 μg of plasmid DNA per 1 × 106 cells and a DNA:polyethylenimine (PEI) ratio of 1:3 (w/w). DNA and the transfection reagent, linear PEI (Polysciences, Inc.), were diluted separately in complete Freestyle 293 media. The DNA was vortexed for 10 min, and DNA and PEI were combined, vortexed for 30 s, allowed to incubate at room temperature for 12 min and finally added to the cell culture. Between 24 and 48 h post transfection, a solution of Tryptone-N1 (Organotechnie SAS) was added to the transfected cell culture at 0.5% (w/v). Cultures were processed on day 5 or 6 post transfection by pelleting and collecting the culture supernatant containing the expressed protein. Protein expression was confirmed by western blot using Strep-Tactin-HRP (IBA). Culture supernatant containing recombinant protein was used in protein overlay assays. For flow cytometry-based-binding assays, PkDBPβ, PkDBPγ and COMP protein were affinity-purified from culture supernatant on a StrepTactin sepharose column (StrepTrap HP; GE Healthcare) using an ÄKTAFPLC Fast Protein Liquid Chromatography system (GE Healthcare). Bound protein was eluted by 2.5 mM desthiobiotin (Sigma-Aldrich). Buffer exchange into PBS pH 7.4 was achieved using centrifugal filter units. Protein concentration was determined from absorbance measurements at 280 nm and the protein-specific extinction coefficient.

Western blot analyses

HEK293-6E culture supernatant, RBCs or RBC ghosts were electrophoresed on 4–15% gradient gels and transferred to nitrocellulose membrane. Membranes were blocked in PBS–0.05% Tween (PBST)–10% milk (for probing with Strep-Tactin-HRP), tris-buffered saline–0.1% Tween–0.5% Neu5Gc-free blocking agent (for probing with anti-Neu5Gc) or PBST–5% milk (for probing with anti-DARC; Sigma-Aldrich). Membranes were probed with the respective antibodies at the indicated dilutions—anti-DARC: 1:1,000 in blocking buffer, anti-Neu5Gc: 1:10,000 in blocking buffer and Strep-Tactin-HRP: 1:2,500 in PBST–20 μg ml−1 avidin (PBST-A) after pre-incubation in PBST-A for 10 min. The DARC western blot was probed with anti-rabbit-HRP (GE Healthcare) at 1:10,000, while the Neu5Gc western blot was probed with anti-chicken/turkey-HRP (Life Technologies) at 1:5,000 in blocking buffer. All membranes were developed with SuperSignal West Pico chemiluminescent substrate (Thermo Scientific) and imaged by autoradiography.

Protein overlay assay

Untreated and enzyme-treated rhesus macaque, and human RBC ghosts and cRBC ghosts were prepared by cell lysis, and multiple washes in cold 5 mM sodium phosphate pH 8.0. Protein content of macaque and human RBC ghosts was quantified by Bradford assay. RBC/cRBC ghosts were stored at −80 °C until use. Before use, cRBC ghosts were treated with DNase I (New England Biolabs). Samples of enzyme-treated or untreated macaque and human RBC ghosts or cRBC ghosts, not treated with reducing agent or heat-denatured, were electrophoresed; 10 μg of macaque or human RBC ghosts (corresponding to ∼3 × 107 cells) were loaded per lane, while cRBC ghosts made from ∼1.7 × 106 cells were loaded per lane. Electrophoresed protein was transferred to nitrocellulose membrane. Membranes were blocked in PBST-10% milk for 2 h at room temperature and then washed in PBST. HEK293-6E culture supernatant containing recombinant protein with Tween 20 added to a final concentration of 0.1% was added to membranes and incubated overnight at 4 °C with gentle rotation. Membranes were washed, blocked in PBST-A for 10 min, incubated in Strep-Tactin-HRP at a dilution of 1:2,500 in PBST-A for 1 h at room temperature and finally developed with Pico substrate. Bound protein was detected by autoradiography.

Flow cytometry-based binding assay

RBCs or cRBCs were washed three times in PBS–0.5% Neu5Gc-free blocking buffer. Cells were pelleted in a 96-well plate at 3 × 105 RBCs per well and resuspended in either 50 μl blocking buffer or PkDBPβ-COMP, PkDBPγ-COMP or COMP protein diluted in blocking buffer at the appropriate concentrations. Samples were incubated for 1 h at room temperature, washed three times in blocking buffer and incubated in a monoclonal anti-strepII-tag antibody (IBA) at 2 μg ml−1 for 1 h at room temperature. Samples were washed three times in blocking buffer then incubated in anti-mouse IgG-Alexa Fluor 488 antibody for 30 min at room temperature. Samples were washed twice, then resuspended in 100 μl PBS for analysis on the MACSQuant flow cytometer. Data were analysed by FlowJo 4 version 10.0.7.

Whole-genome sequencing

Genomic DNA was extracted from schizont-stage Pk H and Pk YH1 parasites using the QIAamp DNA blood kit (Qiagen). Genomic DNA was used to construct Illumina sequencing libraries for both P. knowlesi strains, with a library insert size of 200 bp. Each library was sequenced to a depth of at least 50-fold coverage using 101-bp paired-end reads on an Illumina HiSeq 2000 sequencer. Reads were aligned to the reference genome (P. knowlesi H, PlasmoDB release 11) by applying the bwa mem algorithm from the Burrows–Wheeler Aligner67 to the left and right read files using default settings. Alignments were converted from SAM to BAM format, sorted and indexed using SAMtools68. Coverage was calculated using the BEDtools genomeCoverageBed function69.

Whole-genome sequencing resulted in 23,386,393 reads for Pk H and 21,200,464 reads for Pk YH1. 95.05% of Pk H reads and 88.77% of Pk YH1 reads mapped to the P. knowlesi reference genome. The average coverage depth for Pk H was 89.43% and for Pk YH1 74.59%. These results represent a genome coverage of 99.86% for Pk H and 99.87 for Pk YH1.