Polymorphism in merozoite surface protein-1 gene in north ... - MedIND

Indian J Med Res 130, December 2009, pp 736-741

Polymorphism in merozoite surface protein-1 gene in north & northwest indian field isolates of Plasmodium vivax Umar Farooq, N. Malla & M.L. Dubey

Department of Parasitology, Postgraduate Institute of Medical Education & Research, Chandigarh, India

Received September 19, 2008 Background & objective: Merozoite surface protein-1 of Plasmodium vivax (Pvmsp-1) is a strong vaccine candidate against asexual blood stages. Extensive polymorphism in msp-1 gene has been reported in P. vivax isolates from different geographical regions which is necessary before a field trial of any malaria vaccine based on msp-1 is undertaken. There are only a few reports available on polymorphism in msp-1 gene in Indian field isolates of P. vivax. The aim of the present study was therefore to investigate the polymorphism in Pvmsp-1 gene in 25 isolates of P. vivax collected from malaria patients from regions of north and northwest India. Methods: Parasite DNA was extracted from whole blood samples collected in citrated anticoagulant. The polymorphic region-5, the most variable region of the Pvmsp-1 gene was amplified by PCR. The PCR products were further analyzed by restriction fragment length polymorphism (RFLP) using Mva-1 restriction enzyme. The DNA fragments obtained on PCR and RFLP were analyzed by agarose gel electrophoresis. Results: On the basis of PCR, significant size polymorphism was seen and 4 allelic types were observed amongst the 25 isolates. Further analysis by RFLP discriminated these 4 allelic types into 9 sub-allelic types indicating that PCR-RFLP can be a good tool to study polymorphism in msp-1 gene of Plasmodium. Interpretation & conclusion: Marked genetic polymorphism was observed in msp-1 gene among the isolates of P. vivax. These observations stress the need to study larger numbers of isolates from different regions of India. The findings could have important implications on the vaccine development strategies for P. vivax.

Key words Genetic polymorphism - malaria - MSP-1 - PCR-RFLP - Plasmodium vivax

Though P. falciparum has shown an increased incidence during the last few decades in India, P. vivax continues to be a predominant species contributing around 60 per cent of total cases of malaria in India5. Despite its public health importance, the molecular analysis of P. vivax has been neglected, mainly because the parasite material is

Malaria is an important tropical disease with an estimated global burden of 300 to 660 million cases every year, of which around 90 per cent occur in Sub-Saharan Africa where mortality due to malaria is reported to be higher than elsewhere1,2. Recently Plasmodium vivax has resurged in several countries including Peru and China3,4. 736

Farooq et al: MSP-1 gene polymorphism in Indian P. vivax isolates

limited to small quantities obtained from either infected patients or primate experimental models and unlike P. falciparum, it usually dose not cause severe disease and only rarely causes complicated malaria. The surface of invasive merozoites has been one of the major targets for research to develop an effective malaria vaccine. Merozoite surface protein -1 (msp-1) present on the surface is an important candidate for vaccine development against asexual blood stages6. There is evidence to suggest that MSP-1 can elicit protective immune response against asexual blood stage challenge in animal models6,7. P. vivax msp-1 (Pvmsp-1) is a 200 KDa glycoprotein8 comprising either variable, semi variable or conserved blocks9. The conserved blocks of Pv msp-1 gene show homology with the msp-1 of other Plasmodium species hence these conserved blocks have been designated as interspecies conserved blocks (ICB). The most variable segment of this gene is the one, flanked by interspecies conserved blocks 5 (ICB-5) and 6 and is named as polymorphic block-510. The polymorphic block-5 of Pvmsp-1 gene has been reported to be dimorphic in nature6,7. Two different alleles of this gene namely Belem (type -1) and Salvador-1 (type-2) were identified initially6,7. Later, a third type was identified in Sri Lanka11. The complete gene sequences of the Brazilian Belem (Bel) and Salvador (Sal-1) strains are known. The analysis of specific gene segments derived from parasite isolates from Sri Lanka11, Colombia12, Thailand13 and India5,14 has indicated inter-allelic recombination between the sequences typified as Bel and Sal-1, thus supporting the notion that msp-1 polymorphism in P. vivax is dimorphic in nature, as is in P. falciparum15. Information on polymorphism of msp-1 gene in P. vivax isolates from India is limited. In the present study the extent of polymorphism in msp-1 gene among north and northwest Indian isolates of P. vivax was investigated. Material & Methods Parasite isolates: All patients with P. vivax infection included. Blood samples were collected from symptomatic P. vivax malaria patients (Giemsa stained peripheral blood smear examination) who were attending Nehru Hospital, Postgraduate Institute of Medical Education & Research (PGIMER), Chandigarh, and other different clinics and hospitals in northern India. A total of 25 blood samples were collected

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during January 1999 to December 2001 from different states of northern India (6 from Delhi, 5 from Uttar Pradesh, 4 each from Chandigarh and Haryana, 2 each from Punjab, Rajasthan and Madhya Pradesh). Three patients showing mixed infection (with P. falciparum and P. vivax) were excluded. From each patient, 3-5 ml of blood was obtained by venepuncture in citrate anticoagulant (consisting of 38 mM citric acid, 74 mM sodium citrate and 88 mM sodium chloride) vials16. The study protocol was approved by the Institute Ethics Committee of PGIMER, Chandigarh. Isolation of parasite DNA: Parasite DNA was isolated according to the method described by Foley et al17 with slight modification. Briefly, 50 µl of parasitized blood was washed thrice with 1 ml of ice-cold 5 mM sodium phosphate (pH 8.0) by mixing well by vortexing and centrifuging at 10000 x g for 10 min. The cell pellet was finally resuspended in 50 µl of sterile distilled water and heated in a boiling water bath for 10 min. It was cooled slowly to room temperature, centrifuged at 10000 x g and 40 µl of supernatant was transferred to a new tube for DNA preparation. For PCR, 10 µl of this was used as DNA template in 50 µl PCR mixture. Polymerase chain reaction (PCR): All PCR reactions were carried out in 50 µl reaction volume to amplify block-5 of Pvmsp-1 gene using specific primers12 in a thermal cycler (Master cycler gradient, Eppendorf, Germany). Amplification was performed in 50 mM KCl, 10 mM Tris-Cl (pH 9.0), 1.5 mM MgCl2, 0.01 per cent gelatin, 0.2 mM each of dNTPs, (GIBCO-BRL India Pvt. Ltd, Mumbai, India), 1.25 units of AmpliTaq DNA polymerase (GIBCO-BRL India Pvt. Ltd, Mumbai, India), 250 nM each of sense and antisense primers and 10 µl parasite DNA template. The primers12 used were, 5’ACT ACT TGA TGG TCC TC -3’ and 5’ TTG TGA CAT GCG TAA GCG-3’ (Bangalore Genei Pvt. Ltd, India). The PCR condition were set for initial denaturation at 95oC for 5 min followed by 35 cycles of denaturation at 95oC for 1 min, annealing at 55oC for 2 min and amplification at 72oC for 2 min. The final extension was done at 72oC for 10 min. The DNA extracted from blood samples of P. falciparum infected cases and healthy individuals were used as controls for PCR amplification to check the specificity of the primers. 10 µl of PCR products were run on 1.5 per cent agarose gel with ethidium bromide, visualized under UV transilluminator and photographed. Restriction fragment length polymorphism (RFLP): The amplified product of msp-1 gene block-5 region

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was digested with Mva-1 (CC↓WGG) endonuclease (Fermantas Life Sciences, USA). The selection of the enzyme was done by analyzing published sequences of Pvmsp-1 on website ‘webcutter 2.0’ (http://users. unimi.it/~camelot/tools/cut2.html) and also by using “Gene runner” software programme. Digestion was performed in a 20 µl reaction mixture containing 16 µl of PCR products, 1.5 ml sterile double distilled water, 2 µl 10X digestion buffer and 0.5 µl (5 units) of restriction enzyme. The digestion was carried out at 37oC for overnight. Finally, 10 µl of each digested PCR product was analyzed on a 2 per cent agarose gel, stained with ethidium bromide, visualized under UV transilluminator and photographed. Results All the 25 clinical isolates showed amplification of target gene. No PCR product was obtained in control reactions. PCR amplified products of these isolates showed marked variations in molecular weight ranging from 450 to 550 bp (Fig. 1). On the basis of molecular weight of amplicons, the isolates could be categorized in four allelic types which were designated as type-1 to type-4 (Table I) according to the frequency of the alleles, type-1 being the most frequent and types-3 and

Fig. 1. PCR products and RFLP patterns of MSP-1 gene, polymorphic block-5 (digested with Mva-1) of a few representative P. vivax isolates. Undigested PCR products are included as control for each isolates; digested products were resolved on 2 per cent agarose gel and stained with ethidium bromide. M: Marker-100 bp ladder plus, Lane (L)1: Type-1 allele, L 2: Type-1A sub-allele L 3: Type-1B sub-allele (not digested with Mva-l endonuclease), L 4: Type 2 allele, L 5: Type-2A sub-allele, L 6: Type-2B sub-allele, L7; Type-2C sub-allele, L 8: Type-2D sub-allele (not digested with Mva-l endonuclease), L 9: Type-3 allele, L 10: Type 3 allele, L11: Type 3 allele (undigested control), L 12: type-3B sub allele.

Table I. Allelic types of P. vivax based on molecular weight of PCR products of MSP-1 gene (Polymorphic block-5) (n=25) Approx. size of PCR products (bp) 550 500 450 525 Mixed clone

Allelic type 1 2 3 4 -

No. of P. vivax isolates 8 7 4 4 2

Allelic frequency (%) 32 28 16 16 8

-4 the least frequent (16% each). Two (8%) samples showed two allelic types each indicating mixed clone infection. One was mixture of allelic type-1 and -2 and other of type-3 and -4. Analysis of PCR products by RFLP showed further differences among the allelic types. In each allelic type, the RFLP pattern seen in the majority of isolates was designated as sub allelic type A and those showing other patterns were grouped under sub allelic types B, C or D in order of decreasing frequency. Enzymatic digestion of allelic types-1and -3 produced two distinct sub types (A and B, Fig. 2) while type -2 allele produced 4 subtypes (A, B, C and D, Fig. 1, Table II) Type-4 allele did not produce any sub type (Fig. 2). Interestingly, PCR products of a few isolates could not be digested with Mva-1 restriction enzyme (Fig. 1; Table II).

Fig. 2. RFLP patterns of PCR products of MSP-1 gene, polymorphic block-5 (digested with Mva-1) of a few representative P. vivax isolates; digested products were resolve on 2 per cent agarose gel and stained with ethidium bromide. M: Marker - 100 bp ladder plus, Lane (L)1: PCR products of mixed clone (Types-1 & 2) L2: RFLP pattern of mixed clones (Type-1 & 2), L 3: RFLP pattern of mixed clones (Type-3 & 4), L4 : PCR products of mixed clone (Type-3 & 4), L-5: RFLP pattern of Type-4 allele. L6: PCR product of Type-4 allele.


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Table II. Sub-allelic types of Pvmsp-1 gene (Polymorphic block-5) based on RFLP pattern of PCR products (n=25) Allelic type on the basis of PCR Size (bp)

Allelic type

550

1

500

2

450

3

525 450, 525 (Mixed clone) 550, 500 (Mixed clone)

4 3&4 1&2

Suballelic types on the basis of RFLP Approx. size of digested products (bp) 300 & 250 550 (Not digested) 300 & 200 250 & 250 350 & 150 500 (Not digested) 300 & 150 250 & 200 275 & 250 450 (Not digested) 275 & 250 300 & 250 350 & 150

The RFLP analysis of isolates having mixed clones confirmed the isolates to be mixture of two different clones. The restriction digestion of RJ-6 (isolated and collected from Bikaner, Rajasthan, RJ abbreviation used for Rajasthan, Lane 1 and 2 Fig. 2) isolate (mixture of types-1&2) with Mva-1 produced four fragments of 350, 300, 250 and 150 bp showing the digestion of both the alleles (Fig. 2) while that of CH-3 (isolated from Chandigarh, CH, abbreviation for Chandigarh, Lane 3 and 4, Fig. 2) isolate produced 3 fragments of 450, 275 and 250 bp (Fig. 2) indicating the digestion of one allelic type (type-4) only (Table II). Type 3 clone in this mixed clone isolate was different from sub-allelic types 3A and 3B, as this allele was not digested with Mva1. This sub-allelic type was designated as 3C (Table II). Thus, overall the 25 isolates of P. vivax could be differentiated into 9 genotypes by RFLP analysis. Discussion The use of single copy polymorphic genes like P. vivax msp-1 facilitates identification of heterogeneity in blood stage malaria parasites. In the present study, polymorphic region-5 flanked by interspecies blocks 5 and 6 (ICB 5 and ICB 6) of msp-1 gene was amplified by PCR from total parasite genomic DNA obtained from P. vivax isolates. These isolates showed significant size polymorphism in this region of the gene. This is in agreement with the earlier observation of Putaporntip et al13 in Thai isolates and Maestre et al5 in Indian and Colombian isolates, who have also observed significant size variation in their isolates. In the present study, on the basis of PCR, 4 different genotypes among 25 isolates were observed which confirmed highly polymorphic nature of the

Sub-allelic types

No. of isolates

1A 1B 2A 2B 2C 2D 3A 3B 3C 4 1A 2C

6/8 2/8 3/7 1/7 2/7 1/7 3/4 1/4 4/4 1

Frequency of allele (%) 75 25 42.85 14.28 28.57 14.28 75 25 100 -

1

-

gene. Proto et al18 have observed two types of alleles in Brazilian isolates while three allelic types were observed in Colombian isolates12. In the present study the molecular size of the amplicon of target region of msp-1 of a few isolates of P. vivax (type-3, 450 bp) was close to that of Belem type (454 bp) as reported by Proto et al18 while the size in some isolates (type 4 allele; 525 bp) was close to that of Sal -1 (517 bp) type12,18. Premawansa et al11 have reported the presence of third type of allele in Sri Lankan isolates which had, Sal-1 like block immediately followed by variable numbers of glutamine repeats which were followed by a Belem like sequence indicating that this allele originated as a result of intragenic recombination. In contrast to findings of Maestre et al5 showing absence of Belem type of sequence amongst 9 Indian isolates, in the present study we found both Belem as well as Sal-1 type of alleles amongst 25 isolates. However, we found larger numbers of other allelic types (types 1 & 2), probably due to frequent intragenic recombination in P. vivax. Maestre et al5 also reported the event of intragenic recombination (44%) among Indian and Colombian Isolates. These findings indicate that recombinations are main source of genetic diversity in pvmsp-1 as suggested by other studies11-13,29 . In another study, Putaporntip et al13 observed large size variation in 15 isolates within the same region of the gene and classified these into four types on the basis of sequence analysis. Our study also showed the presence of 4 allelic types on the basis of size of PCR product of same polymorphic region. RFLP analysis of PCR amplified products of this polymorphic region revealed more differences among

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the isolates. With endonuclease Mva-1, 2-4 sub allelic types in three allelic type were obtained. However, PCR amplified product of three isolates belonging to different allele types could not be digested by repeated attempts, suggesting the loss of restriction site for Mva-1 enzyme. Similarly, three alleles of pvmsp-1 gene have been reported from china using PCR-RFLP20. The variation in restriction site for endonuclease within the sequence of different alleles could be generated by intragenic recombination and/or additions or deletions of nucleotides in sequences13. It is believed that the intragenic recombination frequently occur in P. vivax field isolates5,21,22. A number of recombinant alleles have been identified among the Indian and Colombian5, Korean23 and Brazilian19 isolates. Further, Kolakovich et al10 identified that the hybrid sequence of allele showed more than one intragenic recombination. Although several studies have reported higher degree of polymorphism in msp-1 gene in isolates from highly endemic areas but in the present study, isolates originating from areas with low to moderate endemicity also showed marked polymorphism in this gene. Since the exact degree of endemicity of each area from where the isolates were received is not known, the result could not be analyzed according to the degree of endemicity of malaria. Kim et al14 have reported that the P. vivax parasite population is highly diverse in Kolkata, India, despite the low level of transmission. Our results also showed that the use of PCR-RFLP technique to genotype P. vivax isolates on the basis of msp-1 gene might be a powerful tool to identify novel genotypes and could be applied in epidemiological studies and genetic surveys where sequencing is not possible or affordable. The limitation of this method is that it cannot detect some sequence changes like single point mutations, substitution and small deletions which can be detected by sequencing. In conclusion, the study showed that extensive genetic polymorphism exists in msp-1 gene among the north and northwest Indian isolates of P. vivax. These observations stress the need to study larger numbers of isolates from different regions of India. The findings could have important implications on the vaccine development strategies for P. vivax. Acknowledgment Authors thank Dr C.R. Pillai, Emeritus Scientist, National Institute of Malaria Research, Delhi, for providing isolates of P. vivax and acknowledge the Indian Council of Medical Research (ICMR), New Delhi for financial support.

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Reprint requests: Dr M.L. Dubey, Professor, Department of Parasitology, Postgraduate Institute of Medical Education & Research Chandigarh 160 012, India e-mail: [email protected]