Molecular and Biochemical Parasitology, 54 (1992) 121 - 124 121 ((~ 1992 Elsevier Science Publishers B.V. All rights reserved. ,; 0166-6851/92/$05.00

MOLBIO 01796

Short Communication

Second form in a segment of the Merozoite Surface Protein 1 gene of Plasmodium vivax among isolates from Rond6nia (Brazil)

Melissa Po r to a, Marce lo U r b a n o Ferrei ra a, Luiz Marce lo A r a n h a C a m a r g o a, Sunil P r e m a w a n s a b and H e r n a n d o A. del Port i i lo a

"Departamento de Parasitologia, Universidade de Sdo Paulo, Sdo Paulo, Brazil and bDepartrnent of Zoology, Open University of Sri Lanka, Nawala, Sri Lanka

(Received 20 March 1992; accepted I1 May 1992)

Key words: Plasmodium vivax; Merozoite surface protein I; DNA sequence; Polymerase chain reaction; Polymorphism

The Merozoite Surface Protein 1 (MSP-1) of Plasmodiurn species is a high-molecular-weight protein (180-230 kDa) located on the surface of the merozoite and a vaccine candidate against the asexual blood stages of malaria parasites. The complete primary structure of the MSP-1 molecule of Plasmodiurn vivax from the Belem strain (PvMSP-1 BE) [1] and the Sal- 1 strain (PvMSP-I SA) [2] have been reported. The comparison of the deduced amino acid sequences of the MSP-I molecules from the human malarias P. vivax and Plasmodium falciparum (PfMSP-1) and the murine malaria Plasrnodiurn yoelii, revealed the existence of conserved and polymorphic blocks that corre- spond precisely to analogous blocks of several alleles of PfMSP-I [I]. Nucleotide sequence analysis of the polymorphic blocks of several PfMSP-1 alleles revealed that all but one of them could be classed into 2 forms [3-5]. Accordingly, an allele dimorphic model was

Correspondence address." Hernando A. del Portillo, Departa- mento de Parasitologia (ICB II), Av. Lineu Prestes 1374, Universidade de S~o Paulo, Sho Paulo, CEP 05508, Brazil. Tel.: 55-11-813-6944 Ext. 2209. Fax: 55-11-813-0845.

Note: Nucleotide sequence data reported in this paper have been submitted to the GenBank T M data base with the accession number M89629.

proposed to explain the PfMSP-I polymorph- ism [3]. The successful use of the polymerase chain reaction technique (PCR) to study polymorphism of PfMSP-I [6-8] prompted us to try a similar approach to analyze poly- morphism of MSP-1 in P. vivax.

DNA samples from 25 isolates obtained from infected P. vivax patients from the Brazilian Amazon region of Rond6nia were used as PCR substrates using oligomers flanking a polymorphic segment of the PvMSP-1 molecule (poly-Q; diagram, Fig. 1). Twenty-three out of the 25 samples specifically amplified PvMSP-I DNA fragments, as re- vealed by hybridization of Southern blots from all of them with a K2 fragment from the original PvMSP-I BE clone used as a probe (Fig. IA). Significantly, 11/23 (48%) samples amplified 2 different DNA fragments; one of 454 bp, which corresponds to the fragment size generated from the BE clone (lane 1), and one of 517 bp, which may indicate the existence of a second form in this segment of the gene (PvMSP-I RO). To confirm this interpreta- tion, the membranes were washed and re- hybridized with the K2 poly-Q probe from the original BE clone (diagram, Fig. I). None of the 517-bp fragments annealed to this probe, while all of the 454-bp fragments did (Fig. I B).

122

bp

4 5 4 - -

0 S O 0 ( aa )

I I

1 2 3 4 5 6 7 8 9

~"~i-';:;" :" '. ~.:.~.i~'~ .';:-:..:..:_.: ~:>:.;.!~:.;....], .:.:..x-:.:.:~::. [ ,: 62 , _ o 3 1 [ L L L ~ 4 ,_ _c:._%5: t_ ,_(:_~6_,

t poty

K2 Pf H G 7 8 ~ 10 ;~ ~2 13 !4 15 16 17 ~B 19 20 21 22 23 24 25 26 27 28 29 30

• • ~ . . , , . . , . : . . ..: ~ . . . - . . .

A

bp

4S4 - - B

Fig. 1. Second form in a segment of the MSP-I gene of Plasmodium vivax. Genomic DNA (100 200 ng) of 25 samples (lines 6 30) obtained from infected P. vivax patients from Rond6nia, Brazil, was amplified by PCR (94"C/1 min, 42~C/1 min, 72:C/2 min, 25 cycles) using a set of oligomers (*) flanking the Interspecies Conserved Blocks 5 and 6 of the MSP-1 molecule as shown in the diagram. After amplification, a 10-~1 aliquot was analyzed in a 2% agarose gel, blotted and hybridized first with a K2 probe from the original PvMSP-I BE clone (diagram and A) followed by hybridization with BE probe containing only the Poly-Q stretch sequence (diagram and B). As controls, DNA from the PvMSP-1 BE clone (K2), genomic P. falciparum DNA (Pf) and human DNA (H), were amplified with the K2 oligomers, electrophoresed, blotted and hybridized as above. Aftcr hybridizations, all membranes were washed at 2 × SSC for 10 min at room temperature, followed by 3 × 20 min washes in 0.2 × SSC/0.1% SDS at 50°C and exposed overnight at -70'~C to XAR-5 films with intensifying screen• The size in base pairs

(bp) of the K2 PvMSP-I BE fragment is indicated on the left.

The 517-bp PCR product from one sample was cloned and sequenced (Fig. 2). The fragment contains 517 bp encoding a 171-aa polypep- tide, and, except for minor differences, its sequence is identical to the corresponding one from the PvMSP-1 SA gene [2] (Fig. 2). As predicted [I], the amino acid sequences corresponding to the lnterspecies Conserved Blocks 5 and 6 of PvMSP-I were highly conserved with 100% and 98% homologies, respectively; in contrast, a 30% amino acid homology was found within the polymorphic region that flanked them (Fig. 2B). To explore the possibility that the 517 bp DNA fragment

detected in Fig. I A might represent more than one form, a probe from the polymorphic region of the RO clone was generated by digestion with the restriction enzyme TaqI. This probe hybridized to the 517-bp PCR products from all 11 samples (not shown). Two samples (lanes 15 and 23) gave no PCR products with the MSP-1 primers or CS-genc derived control primers [9]. It is thus likely that the parasite infections in these patients were misdiagnosed or the amount of DNA was too low. Since both primer pairs failed to amplify, it is unlikely that these samples contain novel forms of both genes analyzed.

• 70

RO ACTACTTGAT GGTCCTCA.AA AGGGAAATTG ACAAGTTGAA GGACTTCATC CCCAAAATCG AGAGCATGAT SA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . BE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

140

RO CGCCACTGAG AAGAACAAC.-C CGACCGTGGC AC,,CGGCAGAT ATAGTGGCA.A AC.-~ACAATC GCTTAGAGGA SA . . . . . . . . . . . . . . BE . . . . . . . . . . :Tin: . . . . . . ~.A.C.L T:Z61:7: ::::i:Z6: 6i::Z:::i :ZI::Z::

~;0 RO GCAAGTGAAA CAGGGACAAC TGGCAATACA GTCAATGCGC AAACAGCTGT AGTACAACAA CAACAACATC SA . . . . . . . . . . • . . . . . . . . . . C . - ' " BE T . . . . C . . . G C ~1~ G i AC . . . . . . . G T . . . . T . . . A ..AT . . . . . . I I I ~ I . . . A I

ZSO

RO AAGTAGTAAA TC-CAGTA, ACG GTACAGCCTG GAACAACAGG ACATCAAGCA CAAGGTGGAG AAGCAGAA, AC SA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B~ . . - . . . . . . . . . . .AC. . . - . . . . . . . . . . . . . . . . . . - " . . .A. . .CA . . . . C . . . . . . . . . . .AC. . .

3S0

RO ACAJU~AAAT TCAGTACAAG CAC.~AC/U~T TCAACM~ACA CCTGC~G CC,.,G.C,,CGGACA GGTAGCCTCA

SA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . BE .GC . . . . . - . . . . . . . . . .C A.T . . . . . . . AGT..C.G . . . . . . . . . . . . AT.C.CA... A. . .AT . . . .

420

RO ACACAAACGA TTAC.CCA, AGC CCCAC.-CACCA ACTCAAC.-CCT CCCCAGAACC AC-CACCAC.CC G, CCCCACCAT

SA . . . . . . . . . . . . . . . . . . . . • . . . . . . . . . . . . . . . . . . . . . . . et .. . . . . c... C::iZ:i: .c . . . . . . . . ~ i : / ~ ZZ:" . . . . . . . . . . . A.- . . . . . ...r.

• 487

RO CGACAC-TG- TGC-G, CAGTT GCICCAECAC CAACCATGTC CAJU~CTGGAA TATCTG, CIU~A AG, CTCCTTGA SA . . . . . . C..C . . .C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C..CG . . . . . . . . . . . . .

• 490 gO CGACACTGTG CGCAGTTGCT CCAC.-CACCAA CCATGTCCAA ACTGC.AATAT CTGCAAA.AC-C TCCTTGATTT B~ .TG.- . . . . . . . . . . C C . . C . . . . . . . . . G . . . . . . . . . . . . . . . . . . . C . .CG . . . . . . . . . . . . . . . .

517 RO TTTTTTAJUtA TCCG,CTTACG CATGTCACAA SA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

517 RO TTTAAAATCC GCTTACGCAT GTCACAA A BE . . . . . . . . . . . . . . . . . . . . . . . . . . .

I£B5 75 RO YLNVI_ KR[ [ OKI. KOr I pK ] [SJ41 AT~I(NKPT VAJUIOI VAKGQSL RGAS[ TGTTGNT V?4AQTAVV~QQH~VV~iAVT

P H - . . . : : : : i i l l : : : : i l : : i : i : : : i i l : :A. .AAS.P ' - .TS..L . . .$..AA.[VT.I(AVTSE~. • o .Q.- . . . . .

150 RO VOPG T T GHQADGG[A[ T QT NS VQAAQVQQTPAGAGGQVAS TI~ T l SQAPAP T QASP E P APAAPPSTL CA" VAPAP T SA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . PA.A . . . . . . B~ . . . . . . . . .O.QQQ~Q.O(~Q.Q$..VPA...DAQ.. I . . . P T . . S . . . . . .A.GVSATPA.TPAA.-A....A

ICB6 171 gO NSICL E YL ~!CL L DFL KSAYAC H SA . . . . . . . E . . . . . . . . . . . . . B~ . . . . . . . [ . . . . . . . . . . . . . e

Fig. 2. Alignment of the nucleotide (A) and amino acid (B) sequences of a PvMSP-I segment from a Rond6nia isolate (RO) with the analogous segments from the Sal 1 (SA) and Belem (BE) PvMSP-I genes. The 517-bp PCR product from one Rond6nia isolate was cloned into the Bluescript vector (Stratagene) and its sequence was determined from 2 independent colonies by sequencing both strands. The sequences of the oligomers used in the PCR amplifications are underlined and the predicted 11] polymorphic region of these segments shown (*) (A). The location of the ICB5 and ICB6 amino acid blocks as in ref. I is underl ined (B). Dots represent identical nucleotides and lines have been inserted

to optimize the al ignment o f the sequences.

The PCR analysis of P. vivax samples suggests that 2 forms of the PvMSP-! gene exist in the segment amplified among different isolates from Rond6nia (Brazil). Moreover, these 2 forms were frequently (48%) found in a single patient. This finding could indicate that the level of transmission of P. vivax malaria is very high in this Brazilian Amazon region; yet, epidemiological studies revealed a low level of

123

malaria transmission during the month that the samples used in this study were collected (unpublished). An alternative explanation that cannot be excluded using the PvMSP-I BE K2 control used here, is that there are 2 genes of the MSP-I of P. vivax in some parasites.

Although we have only analyzed a small segment of the entire MSP-I gene of P. vivax, its sequence is almost identical to the analo- gous segment of the PvMSP-I SA gene whose primary structure has been completely deter- mined [2]. Therefore, it is reasonable to predict that similarly to the PfMSP-I gene, no more than two major allele forms of the PvMSP-1 gene will be detected when the sample pool analyzed increases. Further experiments are required to confirm our likely conclusion which may gain practical importance for vaccine development against P. vivax in the future.

Acknowledgements

We are particularly grateful to Drs. Ulrich Certa and Alexandra Levitt for critically reading this manuscript, to Professors Erney P. Camargo (WHO Research-based grant 890245) and Luiz Pereira da Silva for encour- agement and support throughout this project; to Drs. Peter H. David and Kamini N. Mendis for the gift of the oligomers; to Dr. Francisco Roberto dos Santos, Director of the Blood Center (HEMERON) at Porto-Velho, Rond6n- ia for kindly providing the lab facilities for the sample collections; and to Maria Jose Menezes for technical assistance. This research received support from the Fundac~lo de Amparo fi Pesquisa do Estado de S~o Paulo (FAPESP) and from the United Nations Development Programme/World Bank/World Health Orga- nization Special programme for Research and Training in Tropical Diseases (TDR).

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