Role of Plasmodium vivax Duffy-binding 1 in invasion of Duffy-null Africans

Karthigayan Gunalana,1, Eugenia Lob,1, Jessica B. Hostetlera,c, Delenasaw Yewhalawd,e, Jianbing Mua, Daniel E. Neafseyf, Guiyun Yanb, and Louis H. Millera,2

aLaboratory of and Vector Research, National Institutes of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD 20852; bProgram in Public Health, College of Health Sciences, University of California, Irvine, CA 92697; cMalaria Programme, Wellcome Trust, Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridge CB10 1RQ, United Kingdom; dDepartment of Medical Laboratory Sciences and Pathology, College of Health Sciences, Jimma University, Jimma 5195, Ethiopia; eTropical and Infectious Diseases Research Center, Jimma University, Jimma 5195, Ethiopia; and fBroad Institute of MIT and Harvard, Cambridge, MA 02142

Contributed by Louis H. Miller, April 18, 2016 (sent for review March 2, 2016; reviewed by John H. Adams and Christopher V. Plowe) The ability of the malaria parasite Plasmodium vivax to invade eryth- binding was determined to be the cysteine-rich region 2 of DBP1 rocytes is dependent on the expression of the Duffy group (9, 10). Later, Duffy blood group was shown to bind antigen on erythrocytes. Consequently, Africans who are null for the DBP1 through a sulfated tyrosine in its first extracellular domain Duffy antigen are not susceptible to P. vivax . Recently, (Fig. S1) (11). P. vivax infections in Duffy-null Africans have been documented, rais- Recently, Duffy-null individuals were found to be infected ing the possibility that P. vivax, a virulent pathogen in other parts of with P. vivax, both throughout Africa (Kenya, Madagascar, P. vivax the world, may expand malarial disease in Africa. binds the Mauritania, Cameroon, Angola, Equatorial Guinea, Ethiopia, Duffy blood group antigen through its Duffy-binding protein 1 (DBP1). and Sudan) and in South America (12–21), raising the question: To determine if mutations in DBP1 resulted in the ability of P. vivax to How can P. vivax invade in the absence of Duffy blood group bind Duffy-null erythrocytes, we analyzed P. vivax parasites obtained expression? Is it possible that mutations in the cysteine-rich re- from two Duffy-null individuals living in Ethiopia where Duffy-null P. vivax and -positive Africans live side-by-side. We determined that, although gion 2 of the DBP1 allows binding to another protein on the DBP1s from these parasites contained unique sequences, they failed the surface of Duffy-null erythrocytes, unrelated to the Duffy? to bind Duffy-null erythrocytes, indicating that mutations in DBP1 did Alternatively, the widespread duplication of the encoding not account for the ability of P. vivax to infect Duffy-null Africans. DBP1 observed in Madagascar (14, 22) and the three and eight However, an unusual DNA expansion of DBP1 (three and eight copies) copies of DBP1 in the two Duffy-null P. vivax-infected patients in in the two Duffy-null P. vivax infections suggests that an expansion of Ethiopia (present paper) may allow for low-affinity binding of DBP1 may have been selected to allow low-affinity binding to another DBP1 to a new receptor on Duffy-null erythrocytes. receptor on Duffy-null erythrocytes. Indeed, we show that Salvador We tested the binding of P. vivax DBP1 in two Duffy-null (Sal) I P. vivax infects Squirrel monkeys independently of DBP1 binding individuals living in Ethiopia to Duffy-positive and -null erythro- to Squirrel monkey erythrocytes. We conclude that P. vivax Sal I and cytes and provide evidence that the former possibility was not the perhaps P. vivax in Duffy-null patients may have adapted to use new case; these DBP1s did not bind Duffy-null erythrocytes. However, – ligand receptor pairs for invasion. concerning the latter possibility, we also provide evidence for DBP1 and DBP2/Erythrocyte-Binding Protein (EBP) (23) independent Plasmodium vivax | Duffy blood group antigen | Duffy-binding protein | of Squirrel monkeys by P. vivax Salvador (Sal) I. DNA expansion Significance n 1975 we identified the failure of Plasmodium knowlesi to invade Duffy-null erythrocytes (1). We assumed that the Duffy I Duffy-null Africans were thought to be resistant to Plasmodium blood group null , a common African phenotype, con- vivax infection. Recently, P. vivax infection was observed in Duffy- ferred resistance in Africans to P. vivax, a Plasmodium closely null Africans. This parasite adaptation is potentially a serious related to P. knowlesi. Subsequent studies demonstrated that Af- public health problem as the majority of African populations are rican American volunteers who were Duffy-null were resistant to Duffy-null. This article is aimed at understanding whether muta- mosquito-transmitted P. vivax (2). In addition, African American tions or DNA expansion in Duffy-binding protein (DBP) contrib- soldiers in Vietnam who were infected with P. vivax were all Duffy- utes to P. vivax Duffy-null infection. Importantly, P. vivax infection positive (3). Furthermore, African Americans in a village in in Squirrel monkey has an ability to use an invasion pathway that MICROBIOLOGY Honduras who were infected with P. vivax were all Duffy-positive is independent of the DBPs. Thus, P. vivax may use a different whereas those infected with P. falciparum were both Duffy-null ligand–receptor pair for its infection in Duffy-null Africans, or and -positive (4). We concluded that Duffy null was the basis of some Duffy-negative Africans are not null but express a low level resistance to P. vivax by Africans. of Duffy blood group antigen. The molecular basis of Duffy null was a single point mutation in the GATA1-binding sequence in the promotor region 5′ to the B – Author contributions: K.G., E.L., G.Y., and L.H.M. designed research; K.G., E.L., D.Y., and J.M. Duffy blood group ORF (Fig. 1 ) that led to Duffy-blood-group performed research; G.Y. led research in Ethiopia; K.G., E.L., J.B.H., D.Y., D.E.N., G.Y., and L.H.M. null erythrocytes (5). In vitro studies with P. knowlesi demon- contributed new reagents/analytic tools; K.G., E.L., J.B.H., D.E.N., G.Y., and L.H.M. analyzed strated that the invasive merozoites were able to bind and reorient data;andK.G.,E.L.,andL.H.M.wrotethepaper. apically with Duffy-null erythrocytes but could not form a junction Reviewers: J.H.A., University of South Florida; and C.V.P., Howard Hughes Medical Institute/ as occurred in Duffy-positive erythrocytes, indicating that the University of Maryland School of Medicine. Duffy blood group was required for P. vivax invasion (6). Later, in The authors declare no conflict of interest. P. knowlesi parasite culture supernatants, the parasite ligand binding Freely available online through the PNAS open access option. to Duffy blood group antigen was identified as the Duffy-binding 1K.G. and E.L. contributed equally to this work. protein 1 (DBP1) (7). Subsequently, P. vivax DBP1 was identified by 2To whom correspondence should be addressed. Email: [email protected]. its ability to bind to Duffy-positive but not to Duffy-null erythrocytes This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. (8). Furthermore the domain within DBP1 that conferred Duffy 1073/pnas.1606113113/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1606113113 PNAS | May 31, 2016 | vol. 113 | no. 22 | 6271–6276 Downloaded by guest on September 24, 2021 Fig. 1. P. vivax infection in Duffy-null Ethiopians. (A) The blood film contains P. vivax ring-stage par- asites in a Duffy-null Ethiopian. (Right) The third panel shows the irregular amoeboid shape of P. vivax in the erythrocyte. (B) GATA1 transcription factor binds to a specific sequence (CTTATCTT) in the upstream pro- moter region of the Duffy blood group antigen. Binding of GATA1 transcription factor is required for the expression of Duffy antigen Fya or Fyb on erythrocytes. A similar GATA1-binding site is observed in both Squirrel and Aotus monkeys’ Duffy blood group antigen. The point mutation at the GATA1- binding site from T to C at position −33 leads to loss of GATA1 binding and results in a homozygous Duffy- null . The heterozygous Duffy blood type individual will have this point mutation (T-33C) in one of the whereas the other is wild type (T −33) and will have less Duffy blood group expression on the erythrocyte surface. (C) Nested PCR amplifica- tion using P. ovale-specific 18S rRNA gene primers. The gel image is representative of two independent experiments. The sample with P. ovale (control MR4- 180) shows a band size of ∼700 bp. In the two Duffy- null samples as well as the remaining control samples, no band was observed for P. ovale.(D) P. vivax-and P. falciparum-specific primers were used in combination in PCR. P. vivax and P. falciparum infection corre- sponds to a band size of 100 and 200 bp, respectively. Both the Duffy-null Ethiopian patients were specifi- cally infected by P. vivax and not by P. falciparum.

Results expressed on COS-7 cells bound Duffy-positive erythrocytes but P. vivax DBP1 Polymorphisms in Binding to Duffy-Null Erythrocytes. failed to bind Duffy-null erythrocytes (Fig. 2 B and C). Our study areas in Ethiopia comprise 65% (129 of 200) Duffy- The DBP1 domains were highly mutated in India VII and positive and 35% (71 of 200) Duffy-null individuals. The P. vivax Brazil I compared with the original P. vivax Sal I sequence. In infections in Duffy-null Ethiopians is less severe than in Duffy- addition to the mutations, the codon for leucine was inserted in positive Ethiopians (20) as was observed in Madagascar (14). these isolates (Fig. 2A), which was unique in the DBP1 se- From the Ethiopian sample collection, we identified two Duffy- quences. Despite the variety of sequences, all bound Duffy- null individuals infected with P. vivax. The blood film from one positive erythrocytes; none bound Duffy-null erythrocytes of the Duffy-null individuals with P. vivax shows ring-stage par- (Fig. 2 B and C). asites confirming the blood-stage infection in this patient (Fig. 1A). These two Duffy-null individuals had homozygous mutation Copy Number Expansion of DBP1 in P. vivax-Infected Duffy-Null (C/C) for Duffy-null in the GATA1-binding region upstream of Patients. Recently, the whole-genome sequencing of P. vivax the ORF (Fig. 1B) and had the ribosomal sequence for P. vivax from a Duffy-positive patient in Madagascar has revealed copy and not for P. falciparum or P. ovale (Fig. 1 C and D). number expansion of Duffy-binding protein 1, and the existence P. vivax The next set of data was the sequence of the DBP1 in of these two DBP1 copies next to each other was confirmed by – Duffy-blood-group null Ethiopians. DBP1s in the two Duffy-null PCR (22). A recent publication describes the gene copy number A patients were different (Fig. 2 ). The two sequences in pRE4 variation of DBP1 in P. vivax from Western Thailand (2 and 3 expressed on the COS-7 surface did not bind Duffy-null copies), Western Cambodia (2 copies), Papua Indonesia (2 and 3 erythrocytes but did bind to Duffy-positive erythrocytes (Fig. 2 B copies) (24). In our study, to minimize potential issues with and C). Despite a unique sequence for the DBP1 from the primer binding because of different boundaries and heterogeneity Duffy-positive Ethiopian sample, the region expressed in pRE4 bound strongly to Duffy-positive erythrocytes, but did not bind to of DBP1 duplication lengths, we performed quantitative real-time Duffy-null erythrocytes (Fig. 2 B and C). PCR targeted to DBP1 and found three and eight copies in the two We tested the P. vivax sequence from Madagascar that was Duffy-null samples from Ethiopia (Fig. 3) compared with the con- found in Duffy-positive and heterozygote Duffy positive/null trols: one copy of PvDBP1 in Sal I (25); one and two copies of individuals (Fig. 2A) (22). The DBP1 from Madagascar was PvDBP1 in Cambodia by whole-genome sequencing (24); and one duplicated, and both sequences were identical in the DBP1 do- copy of aldolase from Sal I. The sample was negative in the no- main except for a single amino acid difference in the signal se- DNA control. In addition, the sequences from each DBP1 from the quence. The Madagascar DBP1 region 2 inserted into pRE4 and two Duffy-null patients were identical in all of the expanded copies

6272 | www.pnas.org/cgi/doi/10.1073/pnas.1606113113 Gunalan et al. Downloaded by guest on September 24, 2021 Fig. 2. Mutations observed in region II of DBP1 from different isolates do not bind Duffy-null erythrocytes. (A) The observed mutations in DBP1 region II in Madagascar (22) and Ethiopia field isolates and in India VII (PVIIG_04680.1) and Brazil I (PVBG_05060.1) strains that were grown in Squirrel monkeys are compared with Sal I (PVX110810). Importantly, Ethiopia Duffy (+)istheP. vivax Duffy-positive sample, and Ethiopia Duffy (−) 1 and 2 are the P. vivax samples from two Duffy-null/negative (−) individuals. The amino acid number for each mutation site is given based on the Salvador I sequence from PlasmoDB. Between position 429 and 430 is an Indel for leucine (L) in the Indian VII and Brazil I sequence. (B) Mutated DBP1 sequences from these isolates were cloned and expressed in COS-7 cells using the pRE4 vector. The binding assay was performed using both Duffy-positive and Duffy-null erythrocytes. The binding MICROBIOLOGY assays show that, regardless of mutations in DBP1, the mutants still bind only Duffy-positive and not Duffy-null erythrocytes. EBP/DBP2 (GenBank: KC987954) binds both Duffy-positive and -null erythrocytes at low frequency. The PfRH5 (PlasmoDB ID: PF3D7_0424100) full-length extracellular domain (64–1578 bp), which is cloned in pRE4 vector, does not bind to either Duffy-positive or -null erythrocytes. In addition, no plasmid-binding control is also plotted. The data are plotted as a log scale on the y axis. At least three independent experiments with replicates were performed. The error bars indicate the SD. (C) Microscopy images show the erythrocyte rosette formation in COS-7 expressing DBP1 from Salvador 1, Ethiopia, Madagascar, India VII, Brazil I, and EBP/DBP2 from Cambodia. Note that the rosettes are smaller in EBP/DBP2. Blue (Hoechst) stain the COS-7 cell nuclei. (Scale bar, 10 μM.)

(sequenced in 32 and 36 DBP1 clones from the two Duffy-null (Fig. 2 B and C). EBP/DBP2 may be a ligand for invasion of Duffy- patients). null erythrocytes.

DBP2 of P. vivax Structurally Related to DBP1 Binds Duffy-Null Invasion of Squirrel Monkey Erythrocytes Is Independent of DBP1 and Erythrocytes. Recently, a new ligand, called EBP/DBP2, was iden- DBP2. It was previously shown that P. vivax infects Squirrel mon- tified in a Cambodian isolate and in other African isolates (23). This keys despite the failure of DBP1 to bind Squirrel erythrocytes protein shares a domain structure similar to DBP1. We expressed (8, 26, 27). The Duffy blood group of Squirrel monkeys has the the protein in COS-7 cells and found that region II of DBP2 binds GATA1-binding domain that expresses the Duffy blood group, both Duffy-positive and Duffy-null erythrocytes at low frequency and previous studies have shown that anti-Fy6, an to a

Gunalan et al. PNAS | May 31, 2016 | vol. 113 | no. 22 | 6273 Downloaded by guest on September 24, 2021 Fig. 3. Copy number expansion in Duffy nulls from Ethiopia. Real-time quantitative PCR plot of relative fluorescent units against the number of threshold cycles (Ct) of P. vivax DBP1 (red) in relation to the single-copy P. vivax aldolase (blue). The green line represents the cutoff where the optimal Ct value of each gene for each sample is determined. The difference in the optimal Ct values (ΔCt; *) between the targeted and reference reflects variation in gene copy number among the samples. The tabular material shows the qPCR data for DBP1 gene copy number for all of the isolates from two independent runs (four replicates each). The double asterisk (**) indicates the DBP1 copy number from the whole-genome sequence (WGS) of the two Cambodian samples (24) and the Sal I parasites (25).

domain upstream of the Duffy-binding region, binds Squirrel the type where the duplication allows for mutations in the second monkey erythrocytes (28) (Fig. 1B and Fig. S1). It is surprising to copy and as a result develops a different function. The six-cys- know that, despite the high similarity between the Duffy blood teine in the P. falciparum family often has duplications, and the group from Squirrel monkeys, Aotus monkeys, and hu- duplicates vary in sequence. For example, Pfs 48/45 is involved in mans (Fig. S1), Sal I P. vivax DBP1 does not bind Squirrel monkey bringing male and female gametes together to assist in fertil- erythrocytes (Fig. 4 A and B). Furthermore, we tested DBP1 from ization, and its duplicate, Pfs 47, permits the ookinetes to pass different P. vivax isolates (India VII and Brazil I) and found that through the mosquito midgut epithelial cells without being DBP1 from India VII and Brazil I formed rosettes with Squirrel marked for destruction by nitrosylation (29). Another example is monkey erythrocytes at the same frequency as Aotus erythrocytes the duplication of CLAG3.1 and 3.2 with the worldwide isolates (Fig. 4 A and B). No duplication of DBP1 occurred in the P. vivax having the identical seven-amino-acid differences between CLAG Sal I (Fig. 3) (25). DBP2, found in the subtelomeric region of 3.1 and 3.2 (30). CLAG 3.1/3.2 have a critical function in Plasmo- chromosome 2, was deleted in P. vivax Sal I. dium spp. in that they form a part of a transporter in the erythrocyte membrane (31). Again there is evidence that they may differ in Discussion function from the mutational differences between the copies (32). Despite different mutations in DBP1 in two Duffy-null Africans The second type of DNA expansion that may be relevant to our in Ethiopia and in Duffy-positive P. vivax infection in different finding involves selective pressure such that the gene protects the parts of the world (Madagascar, India VII, Brazil I, and Sal I), parasite by increasing its expression by DNA expansion. The DNA none can bind Duffy-null erythrocytes when the variable se- expansion can involve multiple genes around the selected gene. One quences are expressed on COS-7 cells, although all bind Duffy- example of this is the introduction of an inhibitor for P. falciparum positive cells (Fig. 2). The basis of DBP1 DNA expansion in dihydroorotate dehydrogenase (DHODH) that leads to DNA Ethiopia is unknown. Studies on DNA expansion in Plasmodium expansion around the gene and always includes a stretch of A’s spp. identify different mechanisms. The simplest to understand is (nucleotides) on each end of the expansion (33). Similar gene

Fig. 4. Salvador I P. vivax infection in the Squirrel monkey is independent of DBP1. (A) Salvador I DBP1 expressed in COS-7 cells does not bind Squirrel mon- key erythrocytes but binds Aotus monkey erythro- cytes. However, DBP1 mutations observed in India VII and Brazil I strains are capable of binding equally to both Squirrel and Aotus monkey erythrocytes. These data indicate that Salvador I infection in the Squirrel monkey is independent of DBP1 and can be a model system to study Duffy-null P. vivax infection that occurs in Africans. The data are plotted as a log scale on the y axis. At least three independent experiments with replicates were performed. The error bars in- dicate the SD. (B) Fluorescent microscopy images show the erythrocyte rosette formation on COS-7 express- ing DBP1 from Salvador I, India VII, and Brazil I using Squirrel and Aotus monkey erythrocytes. Blue (Hoechst) stains the COS-7 cell nuclei. (Scale bar, 10 μM.)

6274 | www.pnas.org/cgi/doi/10.1073/pnas.1606113113 Gunalan et al. Downloaded by guest on September 24, 2021 duplication also occurred in the P. falciparum Multi Drug Resis- Guide for the Care and Use of Laboratory Animals under an animal study tance 1 (MDR1) gene under pressure from mefloquine (34, 35). In proposal approved by the National Institute of Allergy and Infectious Diseases another human infecting parasite Plasmodium knowlesi, duplication Animal Care and Use Committee (NIH). of DBPα, the Duffy blood group binding protein, occurred when An immunofluorescence assay was performed to determine the transfection efficiency using the ID3 antibody that recognized the N terminus of pRE4 and the the parasite was adapted to grow in human erythrocytes (36). DL6 antibody that recognized the extracellular C-terminal end (45). The nuclei The data are limited on the phenotypic significance of increased were stained with Hoechst stain. Immunofluorescence images were taken using DBP1 gene copy numbers in Ethiopia. However, we speculate that 40× objectives from Leica Epi using LASAF software, and the rosettes the increase in copy number may lead to increased messenger RNA were counted using 20× objectives from Carl Zeiss Axiovert 40 CFL microscope. and protein levels. The DNA expansion may facilitate binding to an alternative erythrocyte receptor that has a lower binding affinity to Diagnosis of P. vivax and Duffy Blood Group Antigen Sequencing. Clinical DBP1. The possibility also exists that some Duffy-null individuals samples from Jimma, Ethiopia, were collected to determine P. vivax infection may express a low level of Duffy blood group antigen on their and Duffy blood type. Scientific and ethical clearance was obtained from the erythrocytes despite the inability of GATA1 to bind to the Duffy institutional scientific and ethical review boards of Jimma University, Ethiopia, blood group locus. Three studies have failed to find Duffy blood and the University of California, Irvine. Written informed consent/assent for study participation was obtained from all consenting heads of households, from group antigen on the surface of Duffy-null erythrocytes (14, 37, 38), /guardians (for minors under the age of 18), and from adults for each although extremely low copy number may have been missed. The individual who was willing to participate in the study. For each malaria symp- possibility of low expression of Duffy in P. vivax-infected Duffy-null tomatic or febrile patient, three to four blood spots, equivalent to ∼50 μLeach, individuals has not yet been excluded. were blotted on Whatman 3MM filter paper (Sigma). Parasite DNA was The normal growth of Sal I P. vivax in Squirrel monkeys de- extracted from dried blood spots by the Saponin (Fluka)/Chelex (Bio-Rad) spite the absence of binding of Squirrel monkey erythrocytes to method (46), and genomic DNA was eluted in a total volume of 200 μL TE buffer. DBP1 (Fig. 4) and the deletion of DBP2 raises questions about Slides were examined under using a 100× objective. All slides the receptor used by the Sal I P. vivax on Squirrel monkey eryth- were read in duplicate by two independent microscopists at the time of rocytes. The same receptor may be used for P. vivax invasion of sample collection. A nested amplification of the 18S rRNA gene of Plasmo- dium (P. falciparum, P. vivax, P. malariae, and P. ovale) was performed to Duffy-null erythrocytes. In addition to EBP/DBP2 that binds Duffy- identify positive infection and parasite species using published protocols (47, null erythrocytes, Reticulocyte Homology (RH) genes that are 48). Genomic DNA of each sample was amplified in duplicate for verification. similar in structure to those described in P. falciparum are found in In addition, parasite DNA content was estimated using the SYBR Green qPCR P. vivax (39, 40). P. falciparum RH5 in the RH family was shown to detection method using species-specific primers that targeted the 18S rRNA be critical for invasion of erythrocytes (41), and it is possible that genes (20, 49). All PCR assays included positive controls of both P. falciparum one of the P. vivax RH genes may be involved in invasion of Duffy- 7G8 (MR4-MRA-926) and HB3 (MR4-MRA-155) isolates as well as P. vivax null erythrocytes. There are many other ligands that may explain the Pakchong (MR4-MRA-342G) and Nicaragua (MR4-MRA-340G) isolates (MR4, findings, including those yet to be described. P. vivax is not yet https://www.beiresources.org/About/MR4.aspx, in addition to negative con- adapted to Duffy-null Africans in that it now causes a less severe trols, including uninfected samples and water. For all P. vivax-positive samples, an ∼500-bp fragment of the human Duffy infection than in Duffy-positive people (14, 20). The concern is that − P. vivax – blood group antigen gene that encompasses the 33rd nucleotide position may adapt to infection in Duffy-blood-group null Africans located in the GATA1 transcription factor-binding site of the gene promoter such that it may become a new cause of severe disease (42–44). was amplified and sequenced following published protocols (5, 50). The point mutation T-33C leads to failure of Duffy antigen expression on the surface of Materials and Methods erythrocytes, and individuals with homozygous (C/C) are Duffy-null (5). DBP1 and DBP2 (EBP) Constructs in pRE4 Vector. For the expression of DBP1 mu- tants from different P. vivax strains in COS-7 cells (American Type Culture Col- PCR and Cloning of DBP1 to Identify the Similarities/Differences Between lection), the erythrocyte-binding domain (region II) (10) of different DBP1 Expanded DBP1 Copies. Primers of DBP1 region 2 (forward: 5′-GATATT- mutants [Sal I: PVX_110810, one Ethiopia Duffy-positive and two Ethiopia Duffy- GATCATAAGAAAACGATCTCTAGT-3′; reverse: 5′-TGTCACAACTTCCTGAGT- null patients; India VII: Broad Institute database ID PVIIG_04680.1,BrazilI:Broad ATTTTTTTTAGCCTC-3′) were designed based on the DBP1 region 2 (PVX_110810) Institute database ID PVBG_05060.1 and Madagascar (22)] was synthesized and reference sequence of P. vivax Sal I. Amplification was conducted in a 20-μLre- cloned into the pRE4 vector (kind gift from Gary Cohen and Roselyn Eisenberg, action mixture containing 2 μL of genomic DNA, 10 μLof2×DreamTaqTM Green School of Dental Medicine, University of Pennsylvania, Philadelphia) in unique PCR Master Mix (Fermentas), and 0.5 μM primer. Amplification reactions were ApaI and PvuII restriction enzyme sites. The EBP/DBP2 gene sequence (511– performed in a Bio-Rad MyCycler thermal cycler, with an initial denaturation at 1,452 bp) was also cloned in the pRE4 vector. The EBP/DBP2 full-length se- 94 °C for 2 min, followed by 35 cycles at 94 °C for 30 s, 58 °C for 30 s, and 72 °C for quence is available in GenBank (KC987954). For negative control, the PfRH5 90 s with a final 5-min extension at 72 °C. PCR products were cloned using pDrive (PlasmoDB ID: PF3D7_0424100) gene sequence (64–1,578 bp) without the signal vector (Qiagen). For the two Duffy-negative individuals, 32 and 36 clones were sequence was cloned in the pRE4 vector and does not bind human erythrocytes. sequencedtodeterminethesequence for the DNA expansion. Plasmids were sequenced in both forward and reverse directions using the COS-7 Cells–Erythrocyte-Binding Assay. The assay was performed in an eight- M13 primers (forward: 5′-GTAAAACGACGGCCAGT-3′; reverse: 5′-AACAGC- well chambered cover glass (LAB-TEK, Thermo Fisher Scientific). COS-7 cells TATGACCATG-3′) on ABI 3100 (Applied Biosystems) automated DNA se- MICROBIOLOGY were maintained in DMEM supplemented with 10% (vol/vol) FBS and non- quencer with BigDye dye terminator cycle sequencing kits. Sequences were

essential amino acids at 37 °C with 5% CO2. The day before transfection, the aligned with ClustalX (51) and manually edited in BIOEDIT (52). COS-7 cells (2.5 × 104 cells) were plated on an eight-well chamber. Later the cells were transfected with 200 ng of DBP1-RII from Salvador I, Ethiopia (one Real-Time Quantitative PCR Detection of PvDBP1 Gene Copy Number. We ex- Duffy-positive and two Duffy-null samples), Madagascar, India VII, Brazil I, and amined PvDBP1 DNA expansion in the Ethiopian Duffy-null samples using EBP/DBP2 (Cambodia) using Lipofectamine LTX plus reagent (Invitrogen) primers (forward: 5′-AGGTGGCTTTTGAGAATGAA-3′; reverse: 5′-GAATCT- according to the manufacturer’s protocol. The negative control used in this CCTGGAACCTTCTC-3′) were designed between region II to III of PvDBP1 study is the PfRH5 gene cloned into the pRE4 vector, which did not bind to (PVX_110810) and used along with a reference gene, P. vivax aldolase, which either Duffy-positive or -null erythrocytes. The transfected cells were kept at is known to be a single-copy gene (forward: 5′-GACAGTGCCACCATCCTTACC- 37 °C for 48 h for the surface expression of DBP1-RII, EBP/DBP2, and RH5 in 3′ and reverse: 5′-CCTTCTCAACATTCTCCTTCTTTCC-3′) (53) from P. vivax Sal I transfected COS-7 cells. After 48 h, the transfected COS-7 cells were incubated to estimate gene copy number by the SYBR Green qPCR detection method. with human Duffy-positive and Duffy-null erythrocytes or Squirrel monkey or In addition, two samples from Cambodia, one with a single DBP1 copy and Aotusmonkeyerythrocytes(10%hematocrit)for2hat37°C.Afterin- another sample with two copies determined by whole-genome sequencing cubation, the cells were washed three times with incomplete RPMI. In total, 20 (24), were used as positive control. Water was used as the no-DNA control. or 64 fields were counted for rosette formation from monkey and human Amplification was conducted in a 20-μL reaction mixture containing 2 μLof rosettes, respectively. The blood used in this study was received from the In- genomic DNA, 10 μL2×SYBR Green qPCR Master Mix (Thermo Scientific), and terstate , Memphis, TN, and the NIH blood bank. Monkey eryth- 0.5 μM primer. Reaction was performed in the CFX96 Touch Real-Time PCR rocytes were obtained in accordance with the procedures outlined in the Detection System (Bio-Rad) with an initial denaturation at 95 °C for 3 min,

Gunalan et al. PNAS | May 31, 2016 | vol. 113 | no. 22 | 6275 Downloaded by guest on September 24, 2021 followed by 45 cycles at 94 °C for 30 s, 55 °C for 30 s, and 68 °C for 1 min with a three replicates in the pvdbp1 and pvaldo amplifications, and Scal is an final 95 °C for 10 s. This was then followed by a melting curve step of tem- average SD of the ΔCt values for the calibrator. perature ranging from 65 °C to 95 °C with a 0.5 °C increment to determine the melting temperature of each amplified product. Each assay included an internal ACKNOWLEDGMENTS. We thank Dr. Susan K. Pierce (NIH) for valuable reference gene, the P. vivax aldolase, which is known to be a single copy gene suggestions and critical reading of the manuscript; Mrs. Endalew Zemene, Estifanos Kebede, and Beka Raya (Tropical and Infectious Diseases Research in P. vivax, as well as negative controls (uninfected samples and water). The Center, Jimma University) for sample collection; Drs. Gary Cohen and Roselyn PvDBP1 copy number (n) in each sample was quantified based on the threshold Eisenberg (University of Pennsylvania) for the pRE4 vector and = ΔΔCt±SD ΔΔ = − cycle (Ct) using the follow equation: n 2 ,where Ct (Ctpvaldo (ID3 and DL6); Drs. Thomas E. Wellems, Juliana Sa, and Roberto Moraes Ctpvdbp1) − (Ctpvaldocal − Ctpvdbp1cal). Ctpvaldo and Ctpvdbp1 are threshold cycle Barros for the erythrocytes from Aotus and Squirrel monkeys; Drs. José M. C. Ribeiro, (NIH), Julian C. Rayner (Wellcome Trust Sanger Institute), and Rick values for the aldolase gene and the dbp1 gene, respectively, whereas Ctcal is an average difference between Ct and Ct obtained for the positive con- M. Fairhurst (NIH) for valuable suggestions; and Drs. Lubin Jiang (Institut aldo pvdbp1 Pasteur of Shanghai) and Yang Cheng (NIH) for help in cloning. This work trol Salvador I that contains a single copy of PvDBP1 and aldolase gene was supported by the Intramural Research Program of the Division of Intra- = √ 2 + 2 + 2 fragments. SD is SD calculated as follows: SD (S pvdbp1 S pvaldo S cal), mural Research, National Institute of Allergy and Infectious Diseases, Na- where Spvdbp1 and Spvaldo are the SDs from the average Ct calculated for tional Institutes of Health and Grant R21 AI101802 (to G.Y.).

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