Evolutionary analysis of circumsporozoite surface protein and merozoite surface protein-1 (CSP and MSP-1) sequences of malaria parasites.

Malaria, one of the world's most common diseases, is caused by the intracellular protozoan parasite known as Plasmodium. In this study, we have determined the evolutionary relationship of two single-copy proteins, circumsporozoite protein (CSP) and merozoite surface protein-1 (MSP-1), among Plasmodium species using various bioinformatics tools and softwares. These two proteins are major blood stage antigens of Plasmodium species. This study demonstrates that the circumsporozoite protein of Plasmodium falciparum shows similarity with Plasmodium cynomolgi and Plasmodium knowlesi. The merozoite surface protein-1 of Plasmodium coatneyi forms a monophyletic group with Plasmodium knowlesi, demonstrating their close relationship and these two species also reveal similarity between the human malaria Plasmodium vivax. This Plasmodium phylogenetic arrangement is evidently crucial to identify shared derived characters as well as particular adaptation of plasmodium species from inside and between monophyletic groups.


Background:
Malaria is a common and life-threatening disease in many tropical and subtropical areas. Five species of the plasmodium parasite can infect humans: the most serious forms of the disease are caused by P. falciparum. Malaria caused by P. vivax, P. ovale and P. malariae causes milder disease in humans that is not generally fatal. A fifth species, P. knowlesi, is a zoonosis that causes malaria in macaques but can also infect humans. P. falciparum is the most dangerous of these infections as P. falciparum (or malignant) malaria has the highest rates of complications and mortality. The closest relative of P. falciparum is P. reichenowi, a parasite of chimpanzees. P. falciparum and P. reichenowi are not closely related to the other Plasmodium species that parasitize humans [1,2]. It appears that P. reichenowi was the ancestor of P. falciparum. P. cynomolgi, which infects macaque monkeys, is very closely related to P. vivax and shares the characteristics of hypnozoites, relapse parasitemias, reticulocyte specificity and caveolae vesicular complexes in the infected erythrocyte membrane [1, 3]. The circumsporozoite (CS) protein is the most abundant protein on the surface of malaria sporozoites in the early 1980s [4]. It is a multifunctional molecule that plays a crucial role at various points of the malaria life cycle. This abundant protein forms a dense coat on the circumsporozoite, the form of the parasite that is released into the human bloodstream upon the bite of a mosquito [5]. The single-copy CSP gene encoding the highly immunogenic CSP surface antigen expressed at the parasite's sporozoite stage has proven to be a useful marker for defining the phylogenetic relationship of Plasmodium species [6]. The merozoite surface protein 1 (MSP-1) of Plasmodium plays an important role in erythrocyte invasion by the merozoite and is another potential malaria vaccine candidate antigen [7], which has been characterized from P. vivax, P. cynomolgi, P. knowlesi, P. falciparum and P. yoelii [8,9]. In this study, the evolutionary relationships of two single-copy proteins, circumsporozoite protein and merozoite surface protein-1 of malaria parasite was examined because these proteins are potential malaria vaccine for pre-erythocytic stage. This analysis confirms the CS protein of P. falsiperum and P. reichenowi, P. yoelli and P. berghei, P. coatneyi and P. Knowlesi demonstrates a close relationship and this may also show similar function and structure among them. The MSP-1 protein of P. cynomolgi and P. knowlesi are very much similar and demonstrates close relationship with P. falsiparum.

Methodology:
The protein sequences of CSP and MSP-1 of various malarial parasites were taken from GenBank Database of NCBI (National Center for Biotechnology Information). All the sequences were taken in their FASTA format. These sequences have been listed in Table 1 and Table 2 (see Supplementary material).

Sequence analysis:
Amino acid sequence alignments of these CS and MSP-1 proteins for phylogenetic analysis were generated using Clustal W server [10] and the data obtained was analyzed to determine the similarity between the protein sequences of different Plasmodium species. For multiple sequence alignments, gap open penalty was -7 and gap extension penalty was -1. BLOSUM weight matrix was used for substitution scoring [11,12]. Hydrophilic gap penalties were used to increase the chances of a gap within a run (5 or more residues) of ISSN 0973-2063 (online) 0973-8894 (print) Bioinformation 6(8): 320-323 (2011) 321 © 2011 Biomedical Informatics hydrophilic amino acids; these are likely to be loop or random coil regions where gaps are more common. BioEdit 7.0.2 [13] was also used to perform the protein sequence alignment to determine the number of conserved sites, parsimony info sites, variable sites and singleton sites.

Determination of the Entropy and hydrophobicity:
BioEdit 7.0.2 was used to determine the entropy plot and hydrophobicity values of CS and MSP-1 proteins. From information theory, Entropy can be defined as a measure of the unpredictable nature of a set of possible elements. The higher level of variation within the set, higher the entropy. Entropy is then calculated as given in supplementary material. The information content of a position l, then, is defined as a decrease in uncertainty or entropy at that position. As an alignment improves in quality, the entropy at each position (especially conserved regions) should decrease. This gives a measure of uncertainty at each position relative to other positions. Maximum total uncertainty will be defined by the maximum number of different characters found in a column. The mean hydrophobicity value was plotted for the middle residue of the window. Eisenberg method [14] and Kyte Doolittle method [15] were used to plot hydrophobic moment profile with a window size of 13 residues having six residues on either side of the current residue and rotation angle, θ =100 degrees. (see Supplementary material)

Phylogenetic Analysis:
Phylogenetic and molecular evolutionary analysis was conducted using MEGA version 4.0 [16]. The multiple alignments of sequences of CS and MSP-1 proteins were used to create phylogenetic trees. The evolutionary history was inferred using the Neighbour-Joining method [17]. All the characters were given equal weights. The bootstrap consensus tree inferred from 10000 replicates was taken to represent the evolutionary history of the taxa analyzed [18]. Branches corresponding to partitions reproduced in less than 50% bootstrap replicates were collapsed. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the poisson correction method and are in the units of the number of amino acid substitutions per site. All positions containing gaps and missing data were eliminated from the dataset (Complete deletion option). Phylogenetic analysis was conducted in MEGA4.

Phylogenetic Analysis of CS proteins and MSP-1 proteins:
The phylogenetic trees were constructed by using Neighbour-joining method.
In the case of CS proteins the tree is divided into three different clades ( Figure  5). In the first clade, P. falsiperum shows very much similarity with P. reichenowi and the bootstrap percentage of this similarity is 100. P. yoelli shows similarity with P. berghei in a second clade of this tree with 100 boot strap percentage. But in the third clade P. coatneyi shows very close relationship between P. knowlesi with 100 boot strap percentage and these two malaria parasites show similarity with P. vivax and the boot strap percentage of this similarity is 100. Esmeralda Vargas-Serrato et al. [19] study also shows that these two species (P. coatneyi and P. Knowlesi) are very much similar. The phylogenetic tree of MSP-1 proteins (Figure 6) demonstrates that P. cynomolgi is highly similar with P. knowlesi and the boot strap percentage of this similarity is 83. These two malarial parasites show similarity with P. falsiparum with boot strap percentage of 99. P. yoelli appears with a totally diverged branch from the main tree. The CS and MSP-1 proteins derived phylogenetic trees reveal different topologies, conclusively showing that P. coatneyi and P. knowlesi are evolutionarily closely related and these two malaria parasites show similarity with P. vivax but in case of MSP 1 the P. cynomolgi is highly similar with P. knowlesi and the boot strap percentage of this similarity is 83. P. falsiparum shows very much similarity with these two malarial parasites.

Conclusion:
This study presents a comparative proteomics study and evolutionary analysis of the CS and MSP-1 proteins based on molecular phylogeny across different species of malaria parasite. According to this study we have concluded that CS protein of P. coatneyi and P. knowlesis demonstrate a close relationship with P. vivax and thus it may also show similarity in their structure and function. The MSP-1 protein of P. cynomolgi and P. knowlesi is very much similar and demonstrate close relationship with P. falsiparum.