INTRODUCTION
The rapid gene family expansion in phenotypically important genes indicates that adaptive natural selection favours accumulation of additional copies of genes. The genomes of the Plasmodium spp. present a model system to study lineage specific gene family evolution in response to host-pathogen interaction (duffy binding like and reticulocyte binding protein gene families), pathogenesis and virulence (vir, cir, yir rifin/stevor, var gene families), specific metabolism (proteases, acyl carrier protein, kinases etc.) and the pattern of evolutionary force that leads the evolution of specific biology (Carlton et al. Reference Carlton, Adams, Silva, Bidwell, Lorenzi, Caler, Crabtree, Angiuoli, Merino, Amedeo, Cheng, Coulson, Crabb, Del Portillo, Essien, Feldblyum, Fernandez-Becerra, Gilson, Gueye, Guo, Kang'a, Kooij, Korsinczky, Meyer, Nene, Paulsen, White, Ralph, Ren, Sargeant, Salzberg, Stoeckert, Sullivan, Yamamoto, Hoffman, Wortman, Gardner, Galinski, Barnwell and Fraser-Liggett2008a ). The genome sequences of seven Plasmodium spp. are available in the public domain (Carlton et al. Reference Carlton, Angiuoli, Suh, Kooij, Pertea, Silva, Ermolaeva, Allen, Selengut, Koo, Peterson, Pop, Kosack, Shumway, Bidwell, Shallom, van Aken, Riedmuller, Feldblyum, Cho, Quackenbush, Sedegah, Shoaibi, Cummings, Florens, Yates, Raine, Sinden, Harris, Cunningham, Preiser, Bergman, Vaidya, van Lin, Janse, Waters, Smith, White, Salzberg, Venter, Fraser, Hoffman, Gardner and Carucci2002, Reference Carlton, Adams, Silva, Bidwell, Lorenzi, Caler, Crabtree, Angiuoli, Merino, Amedeo, Cheng, Coulson, Crabb, Del Portillo, Essien, Feldblyum, Fernandez-Becerra, Gilson, Gueye, Guo, Kang'a, Kooij, Korsinczky, Meyer, Nene, Paulsen, White, Ralph, Ren, Sargeant, Salzberg, Stoeckert, Sullivan, Yamamoto, Hoffman, Wortman, Gardner, Galinski, Barnwell and Fraser-Liggett2008a ,Reference Carlton, Escalante, Neafsey and Volkman b ; Gardner et al. Reference Gardner, Hall, Fung, White, Berriman, Hyman, Carlton, Pain, Nelson, Bowman, Paulsen, James, Eisen, Rutherford, Salzberg, Craig, Kyes, Chan, Nene, Shallom, Suh, Peterson, Angiuoli, Pertea, Allen, Selengut, Haft, Mather, Vaidya, Martin, Fairlamb, Fraunholz, Roos, Ralph, McFadden, Cummings, Subramanian, Mungall, Venter, Carucci, Hoffman, Newbold, Davis, Fraser and Barrell2002; Pain et al. Reference Pain, Bohme, Berry, Mungall, Finn, Jackson, Mourier, Mistry, Pasini, Aslett, Balasubrammaniam, Borgwardt, Brooks, Carret, Carver, Cherevach, Chillingworth, Clark, Galinski, Hall, Harper, Harris, Hauser, Ivens, Janssen, Keane, Larke, Lapp, Marti, Moule, Meyer, Ormond, Peters, Sanders, Sanders, Sargeant, Simmonds, Smith, Squares, Thurston, Tivey, Walker, White, Zuiderwijk, Churcher, Quail, Cowman, Turner, Rajandream, Kocken, Thomas, Newbold, Barrell and Berriman2008; Tachibana et al. Reference Tachibana, Sullivan, Kawai, Nakamura, Kim, Goto, Arisue, Palacpac, Honma, Yagi, Tougan, Katakai, Kaneko, Mita, Kita, Yasutomi, Sutton, Shakhbatyan, Horii, Yasunaga, Barnwell, Escalante, Carlton and Tanabe2012) and advances in bioinformatics tools accelerate studies on the evolution of gene families mediating specific malaria parasite phenotypes.
Comparative gene family sequence analysis would provide valuable information about the differential gene family expansion leading to specific biology of malaria parasites. This kind of comparative sequence analysis would also help in identifying unknown/novel members of a gene family from closely related Plasmodium spp. and to understand the pattern of gene family expansion in response to specific biology. In addition, how a gene family is evolving i.e. via gene gain or gene loss or both, can be understood by comparative gene sequence analysis.
At present, complete information about the framework of a vir orthologous gene family in primate malaria parasites other than Plasmodium knowlesi, is yet to be established. The majority of primate malaria parasites are closely related to Plasmodium vivax (Escalante and Ayala, Reference Escalante and Ayala1994, Reference Escalante and Ayala1995; Escalante et al. Reference Escalante, Freeland, Collins and Lal1998) and a vir gene family orthologue among them is unknown. Therefore, identification of a vir orthologous immune evasion gene family from primate malaria parasites would provide valuable information to understand the evolution of virulence in malaria parasites. This study aimed to identify a vir orthologous immune evasion gene family from four primate malaria parasites (Plasmodium cynomolgi, Plasmodium simium, Plasmodium fieldi and Plasmodium simiovale).
MATERIALS AND METHODS
Parasite strains
Four primate malaria parasites viz. P. cynomolgi, P. simium, P. simiovale and P. fieldi were used for identification of a vir-orthologous immune evasion gene family. The genomic DNA of these parasites was obtained from Malaria Research and Reagent Reference Resource Center (MR4), Virginia, USA.
PCR amplification, purification and cloning
We employed a subfamily specific degenerate PCR strategy for amplification of vir gene family orthologues from primate malaria parasites. We have amplified eight subfamilies viz. vir-A, B, C, D, E, G, K and L; PCR primers and amplification protocols used were reported elsewhere (del Portillo et al. Reference del Portillo, Fernandez-Becerra, Bowman, Oliver, Preuss, Sanchez, Schneider, Villalobos, Rajandream, Harris, Pereira da Silva, Barrell and Lanzer2001; Fernandez-Becerra et al. Reference Fernandez-Becerra, Pein, de Oliveira, Yamamoto, Cassola, Rocha, Soares, de Braganca Pereira and del Portillo2005; Carlton et al. Reference Carlton, Adams, Silva, Bidwell, Lorenzi, Caler, Crabtree, Angiuoli, Merino, Amedeo, Cheng, Coulson, Crabb, Del Portillo, Essien, Feldblyum, Fernandez-Becerra, Gilson, Gueye, Guo, Kang'a, Kooij, Korsinczky, Meyer, Nene, Paulsen, White, Ralph, Ren, Sargeant, Salzberg, Stoeckert, Sullivan, Yamamoto, Hoffman, Wortman, Gardner, Galinski, Barnwell and Fraser-Liggett2008a ). The amplified PCR products were purified using a gel extraction kit (MDI, India) and cloned in TA-cloning vector (pTZ257R/T, Fermentas). Positive clones were confirmed by colony PCR method using M13 forward and reverse primers of plasmid. The degenerate primers amplified PCR product is likely to contain multiple sequences, therefore we have cloned the PCR product and sequenced up to 32 positive clones per sample.
Plasmid sequencing and sequence alignment
Plasmid was purified from overnight-grown positive clone bacteria, using a plasmid purification kit (MDI, India). Purified plasmids were sequenced with M13 forward and reverse primers. From a sequenced fragment, vector DNA sequences were trimmed from both 5′ and 3′ primes. Vector free DNA sequences were edited and aligned using DNASTAR Lasergene software version 7.0.0. Each unique sequence was submitted to GenBank.
Sequence identity and phylogenetic analysis
Percentage identity of identified DNA sequences with vir-D subfamily and within primate malaria parasites was done using DNASTAR Lasergene software. DNA sequence of each clone was BLAST at NCBI and Plasmodium genome database (PlasmoDB: www.plasmodb.org). DNA sequences showing identity either with Plasmodium vivax vir gene family or with P. knowlesi kir/sicavar gene family were included for further analysis and those sequences showing identity other than Plasmodium spp. were discarded. A Maximum Likelihood (ML) phylogenetic tree was constructed using newly identified DNA sequences along with vir subfamilies’ specific reference sequences. MEGA software version 5.0 (Kumar et al. Reference Kumar, Nei, Dudley and Tamura2008) was used to construct an ML phylogenetic tree. To understand the topology of the ML phylogenetic tree, 1000 bootstrapping was done.
RESULTS
Degenerate PCR amplification, cloning and sequencing of immune evasion genes
Three subfamilies vir-B, D and G were successfully PCR amplified from P. cynomolgi, P. simiovale and P. fieldi. From P. simiumo only vir-D primer shows amplification. Vir-D amplified PCR product shows a single and expected PCR fragment of 550 bp whereas vir-B and vir-G primers show 800 and 300 bp amplicons respectively along with a few non-specific amplicons. The expected size of PCR product was gel purified, cloned and sequenced. Details of the number of positive clones sequenced are given in Table 1.
Table 1. DNA sequencing of Plasmodium vivax vir gene family orthologues from primate malaria parasites
ND: Not done.
Identification of immune evasion gene family
Vir-B, vir-D and vir-G primers each generated a unique sequence that was represented by an average of three to ten clones (Table 1). These unique sequences were BLAST at NCBI and PlasmoDB to make sure of their identity with malaria parasites. BLAST search result shows that orthologues of vir-D subfamily were present in four primate malaria parasites however the number of unique sequences varied within species (Table 1). A maximum of nine members of a vir-D orthologous immune evasion gene family were identified from P. cynomolgi, four from P. fieldi and one each from P. simium and P. simiovale. Vir-B sequences identified from P. cynomolgi, P. simiovale and P. fieldi show no homology with vir gene family in BLAST search, rather they show homology with other genes in P. vivax and P. knowlesi genomes such as lipoate synthase, lipoyl synthase and hypothetical protein-encoding genes. This suggests that vir-B primer generated sequences from primate malaria parasites do not belong to an immune evasion gene family. Therefore, vir-B primer generated sequences were excluded from further analysis. None of the vir-G primer generated sequences were matched with malaria parasites, rather they show homology with host DNA that suggests contamination or non-specific amplification from host DNA and thus were excluded from the study. We have designated vir orthologous immune evasion gene family as fir for P. fieldi, sir for P. simium and siir for P. simiovale. The immune evasion gene family of P. cynomolgi has already been designated recently as cyir (Tachibana et al. Reference Tachibana, Sullivan, Kawai, Nakamura, Kim, Goto, Arisue, Palacpac, Honma, Yagi, Tougan, Katakai, Kaneko, Mita, Kita, Yasutomi, Sutton, Shakhbatyan, Horii, Yasunaga, Barnwell, Escalante, Carlton and Tanabe2012).
DNA sequence identity of immune evasion gene family
BLAST search confirmed vir orthologous sequences from primate malaria parasites were further analysed to confirm their identity with vir-D subfamily. We did an ML phylogenetic tree construction along with the reference sequences of vir subfamilies (vir-A, B, C, D and E). Interestingly, all sequences obtained from vir-D primer were clustered with vir-D reference sequence (Fig. 1). Since these sequences are orthologous to vir gene family, which is confirmed by BLAST search, therefore in the ML phylogenetic tree a non-vir gene sequence was not used as an out-group. ML phylogeny suggests that the obtained sequences are member of an immune evasion gene family of primate malaria parasites. The genetic identity of these new sequences with vir gene family of P. vivax and between primate malaria parasites is given in Fig. S1. The percentage DNA sequence identity analysis revealed 68–83% identity of primate malaria parasites with P. vivax vir-D sub-family. The percentage DNA sequence identity within members of cyir and fir was 71·2–98·5% and 70·9–71·9% respectively (Fig. S1).
Fig. 1. Phylogenetic tree showing vir-D primer generated DNA sequences from primate malaria parasites are clustered with vir-D subfamily reference sequence. Pcyn: Plasmodium cynomolgi, Pfil: P. fieldi, Psm: P. simium, Psvo: P. simiovale and vir (A-E): Plasmodium vivax vir subfamilies reference sequences. Bootstrapping values are shown in percentage.
DISCUSSION
In this study, we identified orthologues of the P. vivax vir gene family from four primate malaria parasites using a degenerate primer amplification, cloning and DNA sequencing approach. This study shows the presence of a vir orthologous immune evasion gene family in primate malaria parasites and suggests that a vir-D subfamily specific degenerate primer binding site is relatively more conserved among the parasites of the primate malaria clade and may be employed in other primate malaria parasites for the identification of vir orthologous immune evasion gene family.
The identification of vir gene family orthologues from primate malaria parasites was based on the close relationship between P. vivax and parasites of the primate malaria clade (Escalante and Ayala, Reference Escalante and Ayala1994, Reference Escalante and Ayala1995; Escalante et al. Reference Escalante, Freeland, Collins and Lal1998). We assumed a higher degree of sequence homology of vir genes between these species and therefore a degenerate PCR amplification approach was employed. This approach has several limitations. Firstly, vir family members are hypervariable in nature (del Portillo et al. Reference del Portillo, Fernandez-Becerra, Bowman, Oliver, Preuss, Sanchez, Schneider, Villalobos, Rajandream, Harris, Pereira da Silva, Barrell and Lanzer2001; Merino et al. Reference Merino, Fernandez-Becerra, Durham, Ferreira, Tumilasci, d'Arc-Neves, da Silva-Nunes, Ferreira, Wickramarachchi, Udagama-Randeniya, Handunnetti and Del Portillo2006). This may lead to non-specific amplification or no amplification in some cases. The failure of PCR amplification for vir-A, vir-C and vir-E subfamilies and non-specific amplification of vir-B and vir-G could be a possible reason. Secondly, use of vir subfamilies specific degenerate primers provide only a partial fragment of immune evasion genes (del Portillo et al. Reference del Portillo, Fernandez-Becerra, Bowman, Oliver, Preuss, Sanchez, Schneider, Villalobos, Rajandream, Harris, Pereira da Silva, Barrell and Lanzer2001). Thirdly, the degenerate PCR amplification approach may not be able to pinpoint the exact number of members in an immune evasion gene family from closely related species. Therefore, absence of other vir subfamilies members from four primate malaria parasites in the present study reflects experimental limitations and not necessarily the actual absence of these genes.
Antigenic variation in Plasmodium spp. is conferred by a larger repertoire of variant surface proteins which is encoded by an immune evasion gene family (Ferreira et al. Reference Ferreira, da Silva Nunes and Wunderlich2004). The genome sequences of primate malaria clade parasites such as P. vivax (Carlton et al. Reference Carlton, Adams, Silva, Bidwell, Lorenzi, Caler, Crabtree, Angiuoli, Merino, Amedeo, Cheng, Coulson, Crabb, Del Portillo, Essien, Feldblyum, Fernandez-Becerra, Gilson, Gueye, Guo, Kang'a, Kooij, Korsinczky, Meyer, Nene, Paulsen, White, Ralph, Ren, Sargeant, Salzberg, Stoeckert, Sullivan, Yamamoto, Hoffman, Wortman, Gardner, Galinski, Barnwell and Fraser-Liggett2008a ), P. knowlesi (Pain et al. Reference Pain, Bohme, Berry, Mungall, Finn, Jackson, Mourier, Mistry, Pasini, Aslett, Balasubrammaniam, Borgwardt, Brooks, Carret, Carver, Cherevach, Chillingworth, Clark, Galinski, Hall, Harper, Harris, Hauser, Ivens, Janssen, Keane, Larke, Lapp, Marti, Moule, Meyer, Ormond, Peters, Sanders, Sanders, Sargeant, Simmonds, Smith, Squares, Thurston, Tivey, Walker, White, Zuiderwijk, Churcher, Quail, Cowman, Turner, Rajandream, Kocken, Thomas, Newbold, Barrell and Berriman2008) and P. cynomolgi (Tachibana et al. Reference Tachibana, Sullivan, Kawai, Nakamura, Kim, Goto, Arisue, Palacpac, Honma, Yagi, Tougan, Katakai, Kaneko, Mita, Kita, Yasutomi, Sutton, Shakhbatyan, Horii, Yasunaga, Barnwell, Escalante, Carlton and Tanabe2012) show a larger number of members in the immune evasion gene family that may have evolved to shape the specific pathogenesis. Primate malaria parasites are close relatives of one of the most prevalent human malaria parasites, P. vivax, therefore identification of vir orthologous genes from these parasites would provide an important insight into the evolution of virulence among Plasmodium spp. by comparative gene sequence analysis. Although this study has identified a limited number of immune evasion genes from four primate malaria parasites in comparison to the number of members that have been identified in P. vivax (n=∼400), P. knowlesi (n=∼300) and P. cynomolgi (n=∼400), their identification presents a clear picture of relatively higher conservation of vir-D subfamily specific degenerate primer binding sites in primate malaria parasites.
This study clearly identified the presence of vir-D like sequences in four species of Plasmodium infecting primates and suggests that a vir-D subfamily specific primer may be employed for the identification of vir-like genes from other primate malaria parasites. The absence of other vir orthologous members from primate malaria parasites reflects experimental limitation and not necessarily the actual absence of the genes. Further investigation leading to identification of complete members of an immune evasion gene family would be highly helpful in developing deep understanding of how virulence is being evolved among Plasmodium spp. and would be helpful for drug targeting.
CONFLICTS OF INTEREST
The authors declare that they do not have any conflicts of interest.
Supplementary material
To view supplementary material for this article, please visit http://dx.doi.org/S003118201300214X by article doi number.
Acknowledgements
This work was supported by the Indian Council of Medical Research, New Delhi, India (ICMR-Centenary Postdoctoral Fellowship Award). SKP is an ICMR-Centenary Postdoctoral Fellow. The authors would like to thanks MR4 for providing gDNA of primate malaria parasites. This paper bears the NIMR publication screening committee approval no. 021/2013.
