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High level of conservation in Plasmodium vivax merozoite surface protein 4 (PvMSP4)
Infection, Genetics and Evolution 5 (2005) 354–361 www.elsevier.com/locate/meegid
High level of conservation in Plasmodium vivax merozoite surface protein 4 (PvMSP4)$ Pilar Martineza, Carlos F. Suarezb, Andromeda Gomezc, Paula P. Cardenasa, Jose E. Guerreroa, Manuel A. Patarroyoa,c,* a
Molecular Biology Department, Fundacion Instituto de Inmunologia de Colombia, Carrera 50#26-00 Bogota, Colombia b Biomathematics Department, Fundacion Instituto de Inmunologia de Colombia, Carrera 50#26-00 Bogota, Colombia c Universidad Nacional de Colombia, Carrera 30 con Calle 45, Bogota, Colombia Received 11 August 2004; received in revised form 29 October 2004; accepted 1 December 2004 Available online 18 January 2005
Abstract Plasmodium vivax merozoite surface protein 4 (PvMSP4) has been considered to be a promising malarial vaccine candidate. The antigenic diversity displayed by parasite populations is one of the main factors limiting the efficacy of asexual-stage anti-malarial vaccine candidates. The present study is the first characterising PvMSP4 polymorphism. P. vivax isolates were collected from endemic areas in Colombia and diversity and selection pattern studies were carried out. Overall conservation in this protein was remarkable. Changes were only found in exons I and II, the former only having single nucleotide polymorphisms (SNPs) whilst the latter showed variations in tandem repeat number caused by exon II slippage. The remaining regions (EGF-like domain, GPI anchor and intron) were completely conserved. Selection and neutrality tests carried out over variant exons indicated negative selective forces acting on them. No evidence of intragenic recombination was found. The strong conservation displayed in this molecule by isolates from geographically different regions (Colombia, Salvador and Thailand) stresses its potential importance as a candidate for a vaccine against P. vivax malaria. # 2004 Elsevier B.V. All rights reserved. Keywords: Plasmodium vivax; Genetic diversity; Merozoite surface protein 4; Colombian isolates
1. Introduction Malaria is one of the most severe infectious diseases affecting people in developing countries having tropical and subtropical climates. Although Plasmodium vivax is not often fatal for humans, it is a debilitating disease impairing the quality of life and economic productivity. The complex biology of both this parasite and its transmitting vectors has hindered developing successful control strategies. The long$ Nucleotide sequence data reported in this paper are available in the GenBankTM, EMBL and DDBJ databases under the accession numbers: AY707603–AY707622. Abbreviations: EGF, epidermal growth factor; GPI, glycosylphosphatidylinositol; MSP, merozoite surface protein; PCR, polymerase chain reaction; SNP, single nucleotide polymorphism; ASL, adenylosuccinate lyase; bp, base pair * Corresponding author. Tel.: +57 1 3244672x141; fax: +57 1 4815269. E-mail address: [email protected] (M.A. Patarroyo).
1567-1348/$ – see front matter # 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.meegid.2004.12.001
term goal for preventing the disease will require a combined approach through vaccination, chemotherapy and vector control (Fraser et al., 2001). Selected protein antigenic diversity represents one of the major problems in vaccine development. A host response raised against one allele is thus much less effective against parasites expressing different allelic forms (Crewther et al., 1996; Renia et al., 1997). Studying a vaccine candidate’s protein polymorphism therefore becomes a priority when designing vaccines. Analysing polymorphism is also important in establishing the antigenic repertoire of isolates from different endemic regions, enabling antigenic diversity-generating mechanisms to become elucidated. Malarial parasite invasion of erythrocytes is a multi-step process requiring a series of specific molecular interactions. The merozoite represents the invasive form of Plasmodium at the asexual blood-stage. Proteins which are exposed on the merozoite’s surface have been considered as being
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vaccine candidates (Anders and Saul, 2000). Six P. vivax merozoite surface proteins (MSPs) have been identified to date, all being named in accordance with their P. falciparum orthologues (Cui et al., 2003a,b). P. vivax merozoite surface protein 1 (PvMSP1) and P. vivax merozoite surface protein 3a (PvMSP3a) are highly polymorphic and have been used for genotyping P. vivax isolates (del Portillo et al., 1991; Bruce et al., 1999; Galinski et al., 1999; Putaporntip et al., 2002). PvMSP1 variable regions can be grouped into two dimorphic families (types 1 and 2) (del Portillo et al., 1991; Gibson et al., 1992). Evidence of recombination processes generating a third sequence group (type 3) was found some time after the first two (Premawansa et al., 1993). Analysis of polymorphism in several geographical regions has revealed the presence of all three sequence types scattered throughout each region studied (Gutierrez et al., 2000; Lim et al., 2000; Zakeri et al., 2003; Maestre et al., 2004). Sequence analyses have shown single nucleotide polymorphism (SNP) in several blocks creating different alleles as well as variable blocks displaying extensive sequence variation consisting of a number of substitutions, insertions, deletions and a varying number of short tandem repeats. Besides variation induced by selective forces, a strong pattern of intragenic recombination as a new allele-generating mechanism has also been described (Putaporntip et al., 2002). Both size and sequence polymorphism has been previously observed in PvMSP3a alleles (Bruce et al., 1999; Cui et al., 2003a,b). Other studies have shown that PvMSP3a variation (limited to specific domains) has been due to: SNP, slipped-strand mispairing of short DNA repeat units resulting in insertions/deletions and extensive recombination between allelic variants (Rayner et al., 2002). Just two P. vivax strains (Sal-1 and Thai-NYU) have been sequenced to date for the recently identified PvMSP4 (Black et al., 2002), showing that differences in the number of repetitions only lie in the middle of exon I. PvMSP4 is a glycosylphosphatidylinositol (GPI)anchored integral membrane protein possessing an epidermal growth factor (EGF)-like domain at the protein’s carboxyl terminus. It is located downstream of the ASL (adenylosuccinate lyase) gene near msp5, having 301 and 389 bp exons separated by a 156 bp intron. This gene encodes a 230 amino acid protein having 23.3 kDa molecular mass. The N-terminal region has a highly glycine and serine-rich region, displaying the DHGD amino acid motif repeated four times in the Thai-NYU isolate and nine times in the Salvador I strain (Black et al., 2002). PvMSP4 orthologue gene in P. falciparum (PfMSP4), displays a low level of polymorphism consisting of only a few SNPs and three short deletions. PfMSP4 antigenicity has been shown to be strongly conformationally dependent, mainly relying on correct EGF-like domain folding (Wang et al., 1999). The EGF-like domain is less recognised by immunised animals’ sera respecting other protein regions, when presented as a linear epitope (Wang et al., 2001).
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The present study reports the first analysis of PvMSP4 diversity in order to contribute towards assessing this protein’s usefulness as a vaccine candidate.
2. Materials and methods 2.1. Origin of P. vivax samples Blood samples were collected for this study from patients diagnosed as having P. vivax malaria by microscope examination. Samples from 30 patients were extracted, anti-coagulated with EDTA, processed and stored from 1999 to 2002. These samples were obtained from areas to the east and west of the Andes mountain range in Colombia: the Eastern plains (Guaviare, Villavicencio and nearby locations in the Llanos Orientales), the northeastern Department of Norte de Santander (Cu´ cuta and neighbouring areas) and the western Narin˜ o Department (Tumaco and surrounding areas) (Fig. 1A). 2.2. DNA preparation Plasma and leukocytes were removed following centrifugation. DNA was obtained by phenol–chloroform extraction, precipitated in isopropanol, and hydrated in Tris–EDTA buffer for use as template in PCR.
Fig. 1. (A) Geographical location of the regions of study within Colombia. Grey lines on the map represent the areas from which patients came who donated the infected blood samples; the triangles represent sample reception centres; stippled areas represent mountain ranges. (B) The schematic diagram shows P. vivax msp4 gene structure. GPI, glycosylphosphatidylinositol anchor; EGF-like, epidermal growth factor domain with six cystein residues (C).
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2.3. PCR and fragment isolation Two oligonucleotide primers were used for investigating sequence variation in the Pvmsp-4 gene (Fig. 1B): MSP4-D, 50 -GGT GGC CTA CTT TTT GTC-30 and MSP4-R 50 -AAC AAT AAT CAC CAG CAG AAA C-30 , based on GenBank accession AF403475 sequence. The reaction mixture contained 10 mM Tris, 50 mM KCl, 3 mM MgCl2, 20 mM of each dNTP, 1 pmol of each primer, 0.25 U of Taq polymerase and 10–40 ng of DNA template in final 25 ml volume. Thermal profile was as follows: one cycle of 5 min at 95 8C and 30 cycles of 1 min at 95 8C, 30 s at 52.5 8C and 1 min at 72 8C and a final 5 min extension cycle at 72 8C. PCR products were purified with Wizard PCR preps (Promega), ligated into pGEMT-Easy Vector System (Promega) and cloned into E. coli JM109. Positive clones were selected using an ampicillin positive selection system and acomplementation of lacZ gene. Plasmid DNA was extracted by Wizard plus Minipreps (Promega). DNA sequencing was performed by the dideoxynucleotide chain termination method in ABI Prism 310 Automated Sequencer (Applied Biosystems) in both directions using T7 and SP6 primers. Two clones were sequenced per isolate; each one came from an independent PCR amplification. Two extra clones were sequenced to discard errors for those sequences belonging to the same isolate but displaying differences amongst them. P. vivax clinical isolate nucleotide sequences were compared with the previously described Thai-NYU and Sal-1 strain sequences.
the difference between non-synonymous and synonymous substitutions. Positive values for this difference indicate positive selection whilst negative ones indicate negative selection. A t-test assesses the significance of observed differences (Nei and Gojobori, 1986). Tajima’s test (statistic D), also included in DnaSP 3.51 software, was also used. This test is based on neutral model prediction, estimating nucleotide diversity based on the relationship between the number of segregating sites and the average number of nucleotide differences from pairwise comparison. A positive D value indicates possible balancing selection or population subdivision. A negative value suggests recent directional selection, a population bottleneck, or purifying selection (Tajima, 1989). Fu and Li D- and F-tests were also performed; these identify neutrality as a deviation between u estimates (calculated on the number of mutations in external phylogeny branches and from the total number of mutations), giving the D index, or F index from average pairwise diversity p (Fu and Li, 1993). An excess of intermediate frequency polymorphism and a lack of rare variants (singletons) result in positive values for D and F. DnaSP software was used for sliding window analysis for Tajima’s D and Fu and Li’s D- and F-tests to examine whether selective forces might be acting in different directions in several PvMSP4 domains (Rozas and Rozas, 1999).
3. Results
2.4. Sequence analysis
3.1. P. vivax MSP4 genetic polymorphism in Colombian isolates
Colombian P. vivax merozoite surface protein 4 sequences were analysed, as well as a previously reported complete Thai DNA sequence (GenBank accession AF403475) (Black et al., 2002) and partial RNA sequence from Sal-1 strains (GenBank accession AF420240). ClustalX software was used for preliminary sequence alignment and comparing sequences (Thompson et al., 1997). GeneDoc software was used for determining homology and minor editions (Nicholas, 1997). DnaSP 3.51 software was used for calculating p (nucleotide diversity) (Rozas and Rozas, 1999); variant positions having conservative (C) and non-conservative (NC) changes were considered for amino acid sequences. The nature of amino acid replacement (conserved or nonconserved) was examined by using the PAM250 similarity matrix (Dayhoff, 1978). Zero or positive values indicate that variable amino acid residues can be functionally interchanged whilst negative values indicate that it is not common for two amino acids to become replaced in nature. The minimum number of recombination events test (RM), included in DnaSP 3.51 software, was used when searching for evidence of recombination (Hudson and Kaplan, 1985). Natural selection was calculated with MEGA v.2.1 software using the Nei and Gojobori method that compares
Twenty different sequences were obtained from 30 isolates. Only one sequence was obtained per isolate. Two of the isolate sequences were identical to Thai-NYU, both from opposite sides of the Andes Mountains (Tumaco and Norte de Santander). Eleven sequences sorted by identity corresponded to heterogeneous Colombian locations, indicating this protein’s high level of conservation, independently of geographic location. Fig. 2 shows the amino acid alignment for P. vivax MSP4 obtained from Colombian isolates. Sequence analysis presented 16 SNPs (most of them located within exon I) and one parsimonious site in exon II. Four isolates (Lla7, Lla9, Lla12 and Tum4) presented a 12 bp (four amino acids, SSGG at positions 54–57 for ThaiNYU) deletion in the middle of exon I. Exon II presented a different number of short tandem repeats (four and nine times) in the isolates, consisting of 12 bp (GGGGACGATCAC) corresponding to four amino acids DHGD (starting amino acid position 139 in Thai-NYU). Analysis of the nucleotide alignments at this insertion/ deletion suggests that they are being generated by DNA polymerase slippage. In fact, each site having insertion/ deletion is located in a zone of repetitions. Probably, this is a
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Fig. 2. PvMSP4 deduced amino acid sequence alignment, comparing the Sal1 strain, Thai-NYU isolate with Colombian isolates. Asterisk (*) above alignment indicates the position of the codon containing a synonymous mutation. Black letters on a black square represent non-conserved amino acid replacements; black letters on a grey background represent conserved amino acids. Dots indicate conserved residues and dashes represent gaps introduced for alignment. Llanos Orientales, Lla; Norte de Santander, NS; Tumaco, Tum.
diversity-generating mechanism in PvMSP4. Neither the repeat region length, nor the motifs included within it, have shown relationship with the punctual polymorphisms exhibited in other regions of the protein, suggesting that independent mechanisms are generating these variability patterns. The PvMSP4 varies in size between 210 (Thai-NYU, NS11, Tum01) and 230 (Tum02, NS10, NS09, NS08, NS05,
Lla01, Lla02, Lla03, Lla06, Lla08) amino acids, due to the different number of repeats. From a total of 230 amino acid positions considered in the alignment, 10 variable sites (all singletons) were found. Norte de Santander samples possess five of these singleton sites, Llanos samples possess four, while Tumaco sequences only have one of these sites. Most polymorphisms were located in exon I, agreeing with previous reports for P. falciparum (Benet et al., 2004).
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Table 1 Nucleotide diversity for P. falciparum and P. vivax antigens Antigen
Origin
n
Sites
p
Reference
P. vivax
MSP4 MSP1 AMA1 TRAP DBP
Colombia Africa, Asia Africa, Asia Thai, Brazil Colombia
30 175 219 39 23
846 417 461 1470 1388
0.00112 0.0451 0.0174 0.00594 0.01224
This paper Figtree et al. (2000) Figtree et al. (2000) Putaporntip et al. (2001) Martinez et al. (2004)
P. falciparum
MSP1 MSP2 MSP3 AMA1 EBA-175 MSP4
– – Asia – – PNG
40 30 19 9 14 42
1056 417 575 1866 1842 965
0.08792 0.04409 0.09694 0.01635 0.00366 0.0031
Escalante et al. (1998) Escalante et al. (1998) Escalante et al. (1998) Escalante et al. (1998) Escalante et al. (1998) Benet et al. (2004)
Sites: number of sites included; n: number of isolates; p: nucleotide diversity (Escalante et al., 1998; Figtree et al., 2000; Putaporntip et al., 2001; Benet et al., 2004; Martinez et al., 2004).
Intron, GPI anchoring motif, hydrophobic signal sequence and EGF-like domain were completely conserved. 3.2. P. vivax MSP4 sequence analysis Sal-1 and Thai-NYU sequences were employed for cluster analysis, together with Colombian sequences (data not shown); all methods employed resulted in similar tree topology. Non-significant bootstrap values were observed in all trees. This was explained by low PvMSP4 sequence variation. No geographical pattern emerged from cluster analysis, indicating this protein’s high degree of conservation, independently of parasite distribution. DnaSP software was used for finding evidence of recombination but no RM (recombination events) were detected in sequences. Linkage disequilibrium was calculated using D0 as an indicator, but no significant results were obtained (data not shown). Only 10 segregating sites were detected; all changes in exon I were singletons, exon II had only one parsimonious change and four singletons. PvMSP4 showed a low nucleotide diversity (p value = 0.00112). When coding and non-coding regions were analysed, exon I displayed a higher diversity (0.00211), followed by exon II (0.00053), similar to previous P. falciparum MSP4 results (Benet et al., 2004). When sequences from Colombian geographical regions were analysed, Tumaco samples showed less diversity whilst samples obtained from Norte de Santander and the Llanos Orientales had greater diversity. Samples from Villavicencio exhibited the highest number of non-conservative changes (data not shown). Table 1 shows a comparative analysis of nucleotide diversity displayed by several Plasmodium antigens. To date, PvMSP4 shows the lowest nucleotide diversity (0.00112) when compared to other P. vivax or P. falciparum surface proteins. Fig. 2 presents the localisation of conserved and nonconserved changes within sequences. Five synonymous sites were found (replaced in the third position) and almost all
codon replacements occurred in the second base-pair, implicating an amino acid change. More conservative amino acid exchanges than radical changes were observed by contrast with PfMSP4 (Wang et al., 2002) (Fig. 2), suggesting pressure for maintain amino acid properties responding to structural and/or functional constraints. The PvMSP4 protein displayed little sequence variation; this was mainly located in exon I, represented by singleton changes, reflecting that amino terminal region exposure to the immune system may cause diversity pressure. Total sequence conservation was observed for the carboxy terminal region; extreme conservation in both cases suggests functional restriction or recent origin for this parasite’s distribution. 3.3. Evidence of selection in P. vivax MSP4 in Colombian isolates The number of synonymous (dS) and non-synonymous (dN) nucleotide substitutions per site for all pairwise combinations was computed by using the Nei and Gojobori method with Jukes Cantor correction (Table 2). Significant values indicating positive selection (diversifying selection) were obtained for exon I in Colombian isolates and the results maintained the same tendency, irrespective of whether the repeat region was included in the analysis or not. Table 2 Nei–Gojobori test for PvMSP4 Region
Sites
dS
dN
dN dS
S.E.
Exon I Repeat Exon II All
93 36 137 230
0.0000 0.0224 0.0038 0.0020
0.0027 0.0015 0.0005 0.0014
0.0027 0.0209 0.0033 0.0005
0.0009* 0.0143 0.0020 0.0010
Sites: number of sites included; dS: synonymous substitutions; dN: nonsynonymous substitutions; S.E.: standard error. Positive values for dN dS in Nei–Gojobori and Tajima’s test correspond to positive selection pressure and negative values correspond to negative selection pressure. * Significant values according t-test.
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Fig. 3. (A) Sliding-window analysis of Tajima’s and Fu and Li’s tests regarding PvMSP4. The analysis was performed using a 30 nucleotide window size and 15 nucleotide step size. (B) Neutrality tests for PvMSP4 exons I and II. Positive values for Tajima’s and Fu and Li’s tests correspond to diversifying selection pressure and negative values correspond to purifying selection pressure. P > 0.01 significant values are shown underlined and P > 0.02 significant values in italics.
Similar behaviour was found when substitution analysis of coding and non-coding region was done by classifying isolates by geographical origin within Colombia; only samples from Tumaco did not present significant values, but tendency was observed. Most exon II samples presented a tendency to negative selection, while significant positive selection was detected by using the Nei–Gojobori test in exon I. This predominance of non-synonymous polymorphism in exon I suggests that these mutations do not occur at random along the gene and this region may be under diversifying pressure exerted by the host’s immune system. Purifying selection was observed along the entire gene by neutrality tests (Fu and Li’s D* = 3.58610 and F* = 3.67225 and Tajima’s D = 2.26318) (Fig. 3B). These results are the opposite to those obtained with Nei– Gojobori test. These negative values indicate that nucleotide alleles occur with less frequency than expected, many alleles being rare or far from being fixed. DnaSP software was used for sliding window analysis for a neutrality test to analyse these contrasting results and examine those selective forces which might be acting in different directions (Fig. 3A). This revealed negative selection acting in exon I, where singleton mutations were present. A tendency towards positive selection was observed between nucleotides 507 and 522 but was non-significant because mutations were present in intermediate frequencies (Fig. 3A).
These results are consistent with purifying selection action or the generation of recent variants that have not yet become fixed into the population.
4. Discussion Investigations into the extent of sequence variation amongst malaria vaccine candidates are undoubtedly important as the basis of effective malaria vaccine development. This analysis of Colombian isolates has indicated that PvMSP4 polymorphism is manifest by two processes; SNPs generating amino acid replacements (which may be a response to immune pressure) and slipped-strand mispairing of DNA (generating variation in tandem repeat segments); in this case, no relation between slipped strand polymorphism and SNP was found. This is consistent with other reports concerning MSP sequence variation in P. vivax genes such as PvMSP1 (Putaporntip et al., 2002) and PvMSP3 (Rayner et al., 2002). Overall, Colombia is considered to be both a low risk malaria transmission and a low endemic area; the Annual Parasite Index (API) (number of malaria cases per 1000 inhabitants) reported for 2003 was 5.5 (Colombian Ministry of Public Health, 2004, http://www.col.ops-oms.org/sivigila/ 2004/bole08_04.htm). The present study was carried out in Colombian regions having different degrees of endemicity:
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Tumaco (corresponding to the Pacific area, having a 16.74 API in 2003), Llanos Orientales (corresponding to the Orinoquia area, having an 18.10 API in 2003) and Norte de Santander (corresponding to the Center east area, having a 1.32 API in 2003). These API values are considerably lower when compared to high transmission areas (i.e. 293 in Chargaonkala (India) (Subbarao, 2002) or 292 in Portuchuelo (Brazil) (Camargo et al., 1999)). PvMSP4 cluster, diversity and selection results have shown that the Colombian P. vivax populations studied are genetically homogeneous, irrespective of the degree of endemicity; moreover, the high degree of identity amongst samples from Salvador, Thailand and Colombia indicates the low polymorphism that this protein displays throughout the world. According to our previous PvDBP polymorphism studies, we have found a single isolate per patient when samples from the same geographical regions were analysed (Martinez et al., 2004). Polymorphism in PvMSP4 does not occur uniformly throughout the gene; it is remarkable that exon I displayed SNPs, whilst tandem repeat insertion/deletion was preferentially found in exon II. No evidence of recombination was found, unlike that appearing for other antigens such as PvMSP1 and PvMSP3 (Putaporntip et al., 2002; Rayner et al., 2002) becoming the main mechanism for generating allele diversity among isolates from around the globe. Interestingly, PvMSP4 displays the least nucleotide diversity reported in Plasmodium antigens, considering that in previous P. yoelii studies (Kedzierski et al., 2000) it has been shown that this protein is being recognised by the host immune system. It should also be noted that most nucleotide substitutions producing amino acid changes are conservative. Altogether, these findings suggest that conservation in the protein may be necessary for parasite survival. An alternative explanation for high PvMSP4 conservation emerges if the current dispersion of P. vivax around the world has been recent; the high degree of conservation only reflects the high degree of propinquity between Colombian, Central-American and Asian populations. Nevertheless, evidence from other antigens showing greater sequence diversity, accompanied by geographical differentiation, indicates that active host-depending specialisation has been occurring in P. vivax (Putaporntip et al., 2001; Cole-Tobian and King, 2003; Cui et al., 2003a,b; Martinez et al., 2004) and the high conservation in proteins such as PvMSP4 might reflect a true functional restriction. Tests used for estimating the extent of natural selection in PvMSP4 show a predominance of negative selection; however, opposite results were found in exon I, the Nei– Gojobori test showed positive selective pressure and Tajima’s and Fu–Li’s tests showed negative selective pressure. These differences may reflect a different way of estimating variation pattern selection and sensitivity in the sample; nevertheless, other surface antigens analysed in P. vivax have shown the same tendencies (Baum et al., 2003; Cole-Tobian and King, 2003; Martinez et al., 2004),
indicating confrontation between the search for host immune system pressure-mediated sequence-conformational diversity and functional restriction by protein function. The half-life of a protein variant may be short in the population, but play a fundamental role in parasite survival at population level, being fast to adapt to the host’s immune system, with the requirement of maintaining protein functionality. Considering PvMSP4’s high level of conservation and potential role in erythrocyte invasion process, as well as antecedents related to induction and increase of protective immune response in other Plasmodium species, further studies aimed at determining this protein’s antigenic and immunogenic properties will allow the evaluation of its utility as a vaccine component against P. vivax malaria.
Acknowledgements This work was supported by the President of Colombia’s office and the Ministry of Public Health. We would like to thank Stella Buitrago for supplying the P. vivax-infected blood samples and Jason Garry for patiently reviewing the manuscript. We would also like to thank Professor Manuel Elkin Patarroyo for his invaluable comments and suggestions.
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High level of conservation in Plasmodium vivax merozoite surface protein 4 (PvMSP4)$ Pilar Martineza, Carlos F. Suarezb, Andromeda Gomezc, Paula P. Cardenasa, Jose E. Guerreroa, Manuel A. Patarroyoa,c,* a
Molecular Biology Department, Fundacion Instituto de Inmunologia de Colombia, Carrera 50#26-00 Bogota, Colombia b Biomathematics Department, Fundacion Instituto de Inmunologia de Colombia, Carrera 50#26-00 Bogota, Colombia c Universidad Nacional de Colombia, Carrera 30 con Calle 45, Bogota, Colombia Received 11 August 2004; received in revised form 29 October 2004; accepted 1 December 2004 Available online 18 January 2005
Abstract Plasmodium vivax merozoite surface protein 4 (PvMSP4) has been considered to be a promising malarial vaccine candidate. The antigenic diversity displayed by parasite populations is one of the main factors limiting the efficacy of asexual-stage anti-malarial vaccine candidates. The present study is the first characterising PvMSP4 polymorphism. P. vivax isolates were collected from endemic areas in Colombia and diversity and selection pattern studies were carried out. Overall conservation in this protein was remarkable. Changes were only found in exons I and II, the former only having single nucleotide polymorphisms (SNPs) whilst the latter showed variations in tandem repeat number caused by exon II slippage. The remaining regions (EGF-like domain, GPI anchor and intron) were completely conserved. Selection and neutrality tests carried out over variant exons indicated negative selective forces acting on them. No evidence of intragenic recombination was found. The strong conservation displayed in this molecule by isolates from geographically different regions (Colombia, Salvador and Thailand) stresses its potential importance as a candidate for a vaccine against P. vivax malaria. # 2004 Elsevier B.V. All rights reserved. Keywords: Plasmodium vivax; Genetic diversity; Merozoite surface protein 4; Colombian isolates
1. Introduction Malaria is one of the most severe infectious diseases affecting people in developing countries having tropical and subtropical climates. Although Plasmodium vivax is not often fatal for humans, it is a debilitating disease impairing the quality of life and economic productivity. The complex biology of both this parasite and its transmitting vectors has hindered developing successful control strategies. The long$ Nucleotide sequence data reported in this paper are available in the GenBankTM, EMBL and DDBJ databases under the accession numbers: AY707603–AY707622. Abbreviations: EGF, epidermal growth factor; GPI, glycosylphosphatidylinositol; MSP, merozoite surface protein; PCR, polymerase chain reaction; SNP, single nucleotide polymorphism; ASL, adenylosuccinate lyase; bp, base pair * Corresponding author. Tel.: +57 1 3244672x141; fax: +57 1 4815269. E-mail address: [email protected] (M.A. Patarroyo).
1567-1348/$ – see front matter # 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.meegid.2004.12.001
term goal for preventing the disease will require a combined approach through vaccination, chemotherapy and vector control (Fraser et al., 2001). Selected protein antigenic diversity represents one of the major problems in vaccine development. A host response raised against one allele is thus much less effective against parasites expressing different allelic forms (Crewther et al., 1996; Renia et al., 1997). Studying a vaccine candidate’s protein polymorphism therefore becomes a priority when designing vaccines. Analysing polymorphism is also important in establishing the antigenic repertoire of isolates from different endemic regions, enabling antigenic diversity-generating mechanisms to become elucidated. Malarial parasite invasion of erythrocytes is a multi-step process requiring a series of specific molecular interactions. The merozoite represents the invasive form of Plasmodium at the asexual blood-stage. Proteins which are exposed on the merozoite’s surface have been considered as being
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vaccine candidates (Anders and Saul, 2000). Six P. vivax merozoite surface proteins (MSPs) have been identified to date, all being named in accordance with their P. falciparum orthologues (Cui et al., 2003a,b). P. vivax merozoite surface protein 1 (PvMSP1) and P. vivax merozoite surface protein 3a (PvMSP3a) are highly polymorphic and have been used for genotyping P. vivax isolates (del Portillo et al., 1991; Bruce et al., 1999; Galinski et al., 1999; Putaporntip et al., 2002). PvMSP1 variable regions can be grouped into two dimorphic families (types 1 and 2) (del Portillo et al., 1991; Gibson et al., 1992). Evidence of recombination processes generating a third sequence group (type 3) was found some time after the first two (Premawansa et al., 1993). Analysis of polymorphism in several geographical regions has revealed the presence of all three sequence types scattered throughout each region studied (Gutierrez et al., 2000; Lim et al., 2000; Zakeri et al., 2003; Maestre et al., 2004). Sequence analyses have shown single nucleotide polymorphism (SNP) in several blocks creating different alleles as well as variable blocks displaying extensive sequence variation consisting of a number of substitutions, insertions, deletions and a varying number of short tandem repeats. Besides variation induced by selective forces, a strong pattern of intragenic recombination as a new allele-generating mechanism has also been described (Putaporntip et al., 2002). Both size and sequence polymorphism has been previously observed in PvMSP3a alleles (Bruce et al., 1999; Cui et al., 2003a,b). Other studies have shown that PvMSP3a variation (limited to specific domains) has been due to: SNP, slipped-strand mispairing of short DNA repeat units resulting in insertions/deletions and extensive recombination between allelic variants (Rayner et al., 2002). Just two P. vivax strains (Sal-1 and Thai-NYU) have been sequenced to date for the recently identified PvMSP4 (Black et al., 2002), showing that differences in the number of repetitions only lie in the middle of exon I. PvMSP4 is a glycosylphosphatidylinositol (GPI)anchored integral membrane protein possessing an epidermal growth factor (EGF)-like domain at the protein’s carboxyl terminus. It is located downstream of the ASL (adenylosuccinate lyase) gene near msp5, having 301 and 389 bp exons separated by a 156 bp intron. This gene encodes a 230 amino acid protein having 23.3 kDa molecular mass. The N-terminal region has a highly glycine and serine-rich region, displaying the DHGD amino acid motif repeated four times in the Thai-NYU isolate and nine times in the Salvador I strain (Black et al., 2002). PvMSP4 orthologue gene in P. falciparum (PfMSP4), displays a low level of polymorphism consisting of only a few SNPs and three short deletions. PfMSP4 antigenicity has been shown to be strongly conformationally dependent, mainly relying on correct EGF-like domain folding (Wang et al., 1999). The EGF-like domain is less recognised by immunised animals’ sera respecting other protein regions, when presented as a linear epitope (Wang et al., 2001).
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The present study reports the first analysis of PvMSP4 diversity in order to contribute towards assessing this protein’s usefulness as a vaccine candidate.
2. Materials and methods 2.1. Origin of P. vivax samples Blood samples were collected for this study from patients diagnosed as having P. vivax malaria by microscope examination. Samples from 30 patients were extracted, anti-coagulated with EDTA, processed and stored from 1999 to 2002. These samples were obtained from areas to the east and west of the Andes mountain range in Colombia: the Eastern plains (Guaviare, Villavicencio and nearby locations in the Llanos Orientales), the northeastern Department of Norte de Santander (Cu´ cuta and neighbouring areas) and the western Narin˜ o Department (Tumaco and surrounding areas) (Fig. 1A). 2.2. DNA preparation Plasma and leukocytes were removed following centrifugation. DNA was obtained by phenol–chloroform extraction, precipitated in isopropanol, and hydrated in Tris–EDTA buffer for use as template in PCR.
Fig. 1. (A) Geographical location of the regions of study within Colombia. Grey lines on the map represent the areas from which patients came who donated the infected blood samples; the triangles represent sample reception centres; stippled areas represent mountain ranges. (B) The schematic diagram shows P. vivax msp4 gene structure. GPI, glycosylphosphatidylinositol anchor; EGF-like, epidermal growth factor domain with six cystein residues (C).
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2.3. PCR and fragment isolation Two oligonucleotide primers were used for investigating sequence variation in the Pvmsp-4 gene (Fig. 1B): MSP4-D, 50 -GGT GGC CTA CTT TTT GTC-30 and MSP4-R 50 -AAC AAT AAT CAC CAG CAG AAA C-30 , based on GenBank accession AF403475 sequence. The reaction mixture contained 10 mM Tris, 50 mM KCl, 3 mM MgCl2, 20 mM of each dNTP, 1 pmol of each primer, 0.25 U of Taq polymerase and 10–40 ng of DNA template in final 25 ml volume. Thermal profile was as follows: one cycle of 5 min at 95 8C and 30 cycles of 1 min at 95 8C, 30 s at 52.5 8C and 1 min at 72 8C and a final 5 min extension cycle at 72 8C. PCR products were purified with Wizard PCR preps (Promega), ligated into pGEMT-Easy Vector System (Promega) and cloned into E. coli JM109. Positive clones were selected using an ampicillin positive selection system and acomplementation of lacZ gene. Plasmid DNA was extracted by Wizard plus Minipreps (Promega). DNA sequencing was performed by the dideoxynucleotide chain termination method in ABI Prism 310 Automated Sequencer (Applied Biosystems) in both directions using T7 and SP6 primers. Two clones were sequenced per isolate; each one came from an independent PCR amplification. Two extra clones were sequenced to discard errors for those sequences belonging to the same isolate but displaying differences amongst them. P. vivax clinical isolate nucleotide sequences were compared with the previously described Thai-NYU and Sal-1 strain sequences.
the difference between non-synonymous and synonymous substitutions. Positive values for this difference indicate positive selection whilst negative ones indicate negative selection. A t-test assesses the significance of observed differences (Nei and Gojobori, 1986). Tajima’s test (statistic D), also included in DnaSP 3.51 software, was also used. This test is based on neutral model prediction, estimating nucleotide diversity based on the relationship between the number of segregating sites and the average number of nucleotide differences from pairwise comparison. A positive D value indicates possible balancing selection or population subdivision. A negative value suggests recent directional selection, a population bottleneck, or purifying selection (Tajima, 1989). Fu and Li D- and F-tests were also performed; these identify neutrality as a deviation between u estimates (calculated on the number of mutations in external phylogeny branches and from the total number of mutations), giving the D index, or F index from average pairwise diversity p (Fu and Li, 1993). An excess of intermediate frequency polymorphism and a lack of rare variants (singletons) result in positive values for D and F. DnaSP software was used for sliding window analysis for Tajima’s D and Fu and Li’s D- and F-tests to examine whether selective forces might be acting in different directions in several PvMSP4 domains (Rozas and Rozas, 1999).
3. Results
2.4. Sequence analysis
3.1. P. vivax MSP4 genetic polymorphism in Colombian isolates
Colombian P. vivax merozoite surface protein 4 sequences were analysed, as well as a previously reported complete Thai DNA sequence (GenBank accession AF403475) (Black et al., 2002) and partial RNA sequence from Sal-1 strains (GenBank accession AF420240). ClustalX software was used for preliminary sequence alignment and comparing sequences (Thompson et al., 1997). GeneDoc software was used for determining homology and minor editions (Nicholas, 1997). DnaSP 3.51 software was used for calculating p (nucleotide diversity) (Rozas and Rozas, 1999); variant positions having conservative (C) and non-conservative (NC) changes were considered for amino acid sequences. The nature of amino acid replacement (conserved or nonconserved) was examined by using the PAM250 similarity matrix (Dayhoff, 1978). Zero or positive values indicate that variable amino acid residues can be functionally interchanged whilst negative values indicate that it is not common for two amino acids to become replaced in nature. The minimum number of recombination events test (RM), included in DnaSP 3.51 software, was used when searching for evidence of recombination (Hudson and Kaplan, 1985). Natural selection was calculated with MEGA v.2.1 software using the Nei and Gojobori method that compares
Twenty different sequences were obtained from 30 isolates. Only one sequence was obtained per isolate. Two of the isolate sequences were identical to Thai-NYU, both from opposite sides of the Andes Mountains (Tumaco and Norte de Santander). Eleven sequences sorted by identity corresponded to heterogeneous Colombian locations, indicating this protein’s high level of conservation, independently of geographic location. Fig. 2 shows the amino acid alignment for P. vivax MSP4 obtained from Colombian isolates. Sequence analysis presented 16 SNPs (most of them located within exon I) and one parsimonious site in exon II. Four isolates (Lla7, Lla9, Lla12 and Tum4) presented a 12 bp (four amino acids, SSGG at positions 54–57 for ThaiNYU) deletion in the middle of exon I. Exon II presented a different number of short tandem repeats (four and nine times) in the isolates, consisting of 12 bp (GGGGACGATCAC) corresponding to four amino acids DHGD (starting amino acid position 139 in Thai-NYU). Analysis of the nucleotide alignments at this insertion/ deletion suggests that they are being generated by DNA polymerase slippage. In fact, each site having insertion/ deletion is located in a zone of repetitions. Probably, this is a
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Fig. 2. PvMSP4 deduced amino acid sequence alignment, comparing the Sal1 strain, Thai-NYU isolate with Colombian isolates. Asterisk (*) above alignment indicates the position of the codon containing a synonymous mutation. Black letters on a black square represent non-conserved amino acid replacements; black letters on a grey background represent conserved amino acids. Dots indicate conserved residues and dashes represent gaps introduced for alignment. Llanos Orientales, Lla; Norte de Santander, NS; Tumaco, Tum.
diversity-generating mechanism in PvMSP4. Neither the repeat region length, nor the motifs included within it, have shown relationship with the punctual polymorphisms exhibited in other regions of the protein, suggesting that independent mechanisms are generating these variability patterns. The PvMSP4 varies in size between 210 (Thai-NYU, NS11, Tum01) and 230 (Tum02, NS10, NS09, NS08, NS05,
Lla01, Lla02, Lla03, Lla06, Lla08) amino acids, due to the different number of repeats. From a total of 230 amino acid positions considered in the alignment, 10 variable sites (all singletons) were found. Norte de Santander samples possess five of these singleton sites, Llanos samples possess four, while Tumaco sequences only have one of these sites. Most polymorphisms were located in exon I, agreeing with previous reports for P. falciparum (Benet et al., 2004).
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Table 1 Nucleotide diversity for P. falciparum and P. vivax antigens Antigen
Origin
n
Sites
p
Reference
P. vivax
MSP4 MSP1 AMA1 TRAP DBP
Colombia Africa, Asia Africa, Asia Thai, Brazil Colombia
30 175 219 39 23
846 417 461 1470 1388
0.00112 0.0451 0.0174 0.00594 0.01224
This paper Figtree et al. (2000) Figtree et al. (2000) Putaporntip et al. (2001) Martinez et al. (2004)
P. falciparum
MSP1 MSP2 MSP3 AMA1 EBA-175 MSP4
– – Asia – – PNG
40 30 19 9 14 42
1056 417 575 1866 1842 965
0.08792 0.04409 0.09694 0.01635 0.00366 0.0031
Escalante et al. (1998) Escalante et al. (1998) Escalante et al. (1998) Escalante et al. (1998) Escalante et al. (1998) Benet et al. (2004)
Sites: number of sites included; n: number of isolates; p: nucleotide diversity (Escalante et al., 1998; Figtree et al., 2000; Putaporntip et al., 2001; Benet et al., 2004; Martinez et al., 2004).
Intron, GPI anchoring motif, hydrophobic signal sequence and EGF-like domain were completely conserved. 3.2. P. vivax MSP4 sequence analysis Sal-1 and Thai-NYU sequences were employed for cluster analysis, together with Colombian sequences (data not shown); all methods employed resulted in similar tree topology. Non-significant bootstrap values were observed in all trees. This was explained by low PvMSP4 sequence variation. No geographical pattern emerged from cluster analysis, indicating this protein’s high degree of conservation, independently of parasite distribution. DnaSP software was used for finding evidence of recombination but no RM (recombination events) were detected in sequences. Linkage disequilibrium was calculated using D0 as an indicator, but no significant results were obtained (data not shown). Only 10 segregating sites were detected; all changes in exon I were singletons, exon II had only one parsimonious change and four singletons. PvMSP4 showed a low nucleotide diversity (p value = 0.00112). When coding and non-coding regions were analysed, exon I displayed a higher diversity (0.00211), followed by exon II (0.00053), similar to previous P. falciparum MSP4 results (Benet et al., 2004). When sequences from Colombian geographical regions were analysed, Tumaco samples showed less diversity whilst samples obtained from Norte de Santander and the Llanos Orientales had greater diversity. Samples from Villavicencio exhibited the highest number of non-conservative changes (data not shown). Table 1 shows a comparative analysis of nucleotide diversity displayed by several Plasmodium antigens. To date, PvMSP4 shows the lowest nucleotide diversity (0.00112) when compared to other P. vivax or P. falciparum surface proteins. Fig. 2 presents the localisation of conserved and nonconserved changes within sequences. Five synonymous sites were found (replaced in the third position) and almost all
codon replacements occurred in the second base-pair, implicating an amino acid change. More conservative amino acid exchanges than radical changes were observed by contrast with PfMSP4 (Wang et al., 2002) (Fig. 2), suggesting pressure for maintain amino acid properties responding to structural and/or functional constraints. The PvMSP4 protein displayed little sequence variation; this was mainly located in exon I, represented by singleton changes, reflecting that amino terminal region exposure to the immune system may cause diversity pressure. Total sequence conservation was observed for the carboxy terminal region; extreme conservation in both cases suggests functional restriction or recent origin for this parasite’s distribution. 3.3. Evidence of selection in P. vivax MSP4 in Colombian isolates The number of synonymous (dS) and non-synonymous (dN) nucleotide substitutions per site for all pairwise combinations was computed by using the Nei and Gojobori method with Jukes Cantor correction (Table 2). Significant values indicating positive selection (diversifying selection) were obtained for exon I in Colombian isolates and the results maintained the same tendency, irrespective of whether the repeat region was included in the analysis or not. Table 2 Nei–Gojobori test for PvMSP4 Region
Sites
dS
dN
dN dS
S.E.
Exon I Repeat Exon II All
93 36 137 230
0.0000 0.0224 0.0038 0.0020
0.0027 0.0015 0.0005 0.0014
0.0027 0.0209 0.0033 0.0005
0.0009* 0.0143 0.0020 0.0010
Sites: number of sites included; dS: synonymous substitutions; dN: nonsynonymous substitutions; S.E.: standard error. Positive values for dN dS in Nei–Gojobori and Tajima’s test correspond to positive selection pressure and negative values correspond to negative selection pressure. * Significant values according t-test.
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Fig. 3. (A) Sliding-window analysis of Tajima’s and Fu and Li’s tests regarding PvMSP4. The analysis was performed using a 30 nucleotide window size and 15 nucleotide step size. (B) Neutrality tests for PvMSP4 exons I and II. Positive values for Tajima’s and Fu and Li’s tests correspond to diversifying selection pressure and negative values correspond to purifying selection pressure. P > 0.01 significant values are shown underlined and P > 0.02 significant values in italics.
Similar behaviour was found when substitution analysis of coding and non-coding region was done by classifying isolates by geographical origin within Colombia; only samples from Tumaco did not present significant values, but tendency was observed. Most exon II samples presented a tendency to negative selection, while significant positive selection was detected by using the Nei–Gojobori test in exon I. This predominance of non-synonymous polymorphism in exon I suggests that these mutations do not occur at random along the gene and this region may be under diversifying pressure exerted by the host’s immune system. Purifying selection was observed along the entire gene by neutrality tests (Fu and Li’s D* = 3.58610 and F* = 3.67225 and Tajima’s D = 2.26318) (Fig. 3B). These results are the opposite to those obtained with Nei– Gojobori test. These negative values indicate that nucleotide alleles occur with less frequency than expected, many alleles being rare or far from being fixed. DnaSP software was used for sliding window analysis for a neutrality test to analyse these contrasting results and examine those selective forces which might be acting in different directions (Fig. 3A). This revealed negative selection acting in exon I, where singleton mutations were present. A tendency towards positive selection was observed between nucleotides 507 and 522 but was non-significant because mutations were present in intermediate frequencies (Fig. 3A).
These results are consistent with purifying selection action or the generation of recent variants that have not yet become fixed into the population.
4. Discussion Investigations into the extent of sequence variation amongst malaria vaccine candidates are undoubtedly important as the basis of effective malaria vaccine development. This analysis of Colombian isolates has indicated that PvMSP4 polymorphism is manifest by two processes; SNPs generating amino acid replacements (which may be a response to immune pressure) and slipped-strand mispairing of DNA (generating variation in tandem repeat segments); in this case, no relation between slipped strand polymorphism and SNP was found. This is consistent with other reports concerning MSP sequence variation in P. vivax genes such as PvMSP1 (Putaporntip et al., 2002) and PvMSP3 (Rayner et al., 2002). Overall, Colombia is considered to be both a low risk malaria transmission and a low endemic area; the Annual Parasite Index (API) (number of malaria cases per 1000 inhabitants) reported for 2003 was 5.5 (Colombian Ministry of Public Health, 2004, http://www.col.ops-oms.org/sivigila/ 2004/bole08_04.htm). The present study was carried out in Colombian regions having different degrees of endemicity:
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Tumaco (corresponding to the Pacific area, having a 16.74 API in 2003), Llanos Orientales (corresponding to the Orinoquia area, having an 18.10 API in 2003) and Norte de Santander (corresponding to the Center east area, having a 1.32 API in 2003). These API values are considerably lower when compared to high transmission areas (i.e. 293 in Chargaonkala (India) (Subbarao, 2002) or 292 in Portuchuelo (Brazil) (Camargo et al., 1999)). PvMSP4 cluster, diversity and selection results have shown that the Colombian P. vivax populations studied are genetically homogeneous, irrespective of the degree of endemicity; moreover, the high degree of identity amongst samples from Salvador, Thailand and Colombia indicates the low polymorphism that this protein displays throughout the world. According to our previous PvDBP polymorphism studies, we have found a single isolate per patient when samples from the same geographical regions were analysed (Martinez et al., 2004). Polymorphism in PvMSP4 does not occur uniformly throughout the gene; it is remarkable that exon I displayed SNPs, whilst tandem repeat insertion/deletion was preferentially found in exon II. No evidence of recombination was found, unlike that appearing for other antigens such as PvMSP1 and PvMSP3 (Putaporntip et al., 2002; Rayner et al., 2002) becoming the main mechanism for generating allele diversity among isolates from around the globe. Interestingly, PvMSP4 displays the least nucleotide diversity reported in Plasmodium antigens, considering that in previous P. yoelii studies (Kedzierski et al., 2000) it has been shown that this protein is being recognised by the host immune system. It should also be noted that most nucleotide substitutions producing amino acid changes are conservative. Altogether, these findings suggest that conservation in the protein may be necessary for parasite survival. An alternative explanation for high PvMSP4 conservation emerges if the current dispersion of P. vivax around the world has been recent; the high degree of conservation only reflects the high degree of propinquity between Colombian, Central-American and Asian populations. Nevertheless, evidence from other antigens showing greater sequence diversity, accompanied by geographical differentiation, indicates that active host-depending specialisation has been occurring in P. vivax (Putaporntip et al., 2001; Cole-Tobian and King, 2003; Cui et al., 2003a,b; Martinez et al., 2004) and the high conservation in proteins such as PvMSP4 might reflect a true functional restriction. Tests used for estimating the extent of natural selection in PvMSP4 show a predominance of negative selection; however, opposite results were found in exon I, the Nei– Gojobori test showed positive selective pressure and Tajima’s and Fu–Li’s tests showed negative selective pressure. These differences may reflect a different way of estimating variation pattern selection and sensitivity in the sample; nevertheless, other surface antigens analysed in P. vivax have shown the same tendencies (Baum et al., 2003; Cole-Tobian and King, 2003; Martinez et al., 2004),
indicating confrontation between the search for host immune system pressure-mediated sequence-conformational diversity and functional restriction by protein function. The half-life of a protein variant may be short in the population, but play a fundamental role in parasite survival at population level, being fast to adapt to the host’s immune system, with the requirement of maintaining protein functionality. Considering PvMSP4’s high level of conservation and potential role in erythrocyte invasion process, as well as antecedents related to induction and increase of protective immune response in other Plasmodium species, further studies aimed at determining this protein’s antigenic and immunogenic properties will allow the evaluation of its utility as a vaccine component against P. vivax malaria.
Acknowledgements This work was supported by the President of Colombia’s office and the Ministry of Public Health. We would like to thank Stella Buitrago for supplying the P. vivax-infected blood samples and Jason Garry for patiently reviewing the manuscript. We would also like to thank Professor Manuel Elkin Patarroyo for his invaluable comments and suggestions.
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