Natural selection and population genetic structure of domain-I of Plasmodium falciparum apical membrane antigen-1 in India
Graphical abstract
Introduction
Malaria is a serious public health problem in the tropics with estimated 216 million cases and 655,000 deaths in 2010 (World Health Organization., 2011) Immunization with AMA-1 induces antibodies that inhibit invasion, conferring protection in animals, mostly due to infections of the most virulent human malaria parasite, Plasmodium falciparum (Genton and Reed, 2007, Hu et al., 2008, Rodrigues et al., 2005). For effective control of this deadly disease, vaccines are decisively needed. Immunization with different blood-stage antigens is shown to be protective in a number of animal models (Malkin et al., 2005, Narum et al., 2000, Stowers et al., 2002). At present, the leading blood-stage vaccine candidates are all proteins expressed during the invasion of the red blood cells (RBCs), either contained within the apical organelles or located on the merozoite surface (Alaro et al., 2010). One of the current challenges in developing vaccines targeting these antigens is the high level of genetic diversity among Plasmodium isolates in different geographical areas across the world. Highly polymorphic regions have been observed in P. falciparum and Plasmodium vivax antigenic surface proteins such as Circumsporozoite protein (CSP), Duffy-binding protein (DBP), Merozoite surface protein-1 (MSP-1), Apical membrane antigen-1 (AMA-1) and Thrombospondin related anonymous protein (TRAP) (Chenet et al., 2012). Furthermore, the polymorphic regions are not evenly distributed across these antigenic proteins (Escalante et al., 1998, Franks et al., 2003, Mu et al., 2007, Polley and Conway, 2001, Volkman et al., 2007).
Protein–protein interactions (PPIs) serve as the central mechanism in most cellular functions and it is essentially guided by amino acid residues with different structural and functional constraints (Bonsor and Sundberg, 2011). Mutations in these contact residues may cause altered binding properties and thus different amino acids will be subject to position specific selective pressures (Hoberman et al., 2004). Until recently most protein–protein interactions have been proved experimentally at the single amino acid level making them capable of capturing the combinatorial effect of mutagenesis and selection at population level (Munz et al., 2012, Nannemann et al., 2011). In this investigation, we study the genetic diversity of AMA-1 not only to evaluate the processes maintaining the variation but in addition to explain functionally the nature and pattern of amino acid substitutions observed in parasite population.
The severe pathophysiological manifestations of malaria caused by P. falciparum are a direct consequence of the parasite’s blood stage replication cycle, during which merozoites repeatedly invade, multiply within, and destroy red blood cells (Woehlbier et al., 2010). A number of parasite proteins have been implicated in RBC invasion that are comprised of (a) GPI-Anchored MSP family proteins (MSP1/2/4/10), (b) microneme proteins (AMA1, EBA-175, EBA-140/BAEBL, EBA-181/JESEBL), (c) peripheral surface proteins (MSP3/6/7, SERA3/4/5/6) and (d) rhoptry neck proteins (RON2/4 etc.) (Cowman and Crabb, 2006). The exposure of P. falciparum merozoites to low potassium ion concentrations as found in blood plasma leads to a rise in cytosolic calcium levels which triggers secretion of 175 kD erythrocyte binding antigen (EBA175) and apical membrane antigen-1 (AMA-1) to the merozoite surface (Singh et al., 2010). Subsequent interaction of EBA175 with glycophorin A (glyA), its receptor on erythrocytes, restores basal cytosolic calcium levels and triggers release a set of rhoptry neck-derived parasite proteins (RON proteins) which associate with PfAMA1 at the moving junction, a step crucial for RBC invasion (Alexander et al., 2006, Collins et al., 2009, Dutta et al., 2003, Lamarque et al., 2011, Tonkin et al., 2011). It has been demonstrated that antibodies directed against AMA-1 at the time of presentation with malaria are strongly associated with concurrent and future blood stage immunity, as measured by the ability of the host to clear P. falciparum (Keh et al., 2012). AMA-1 recombinant vaccines based on P. falciparum 3D7 and FVO strain have shown an excellent efficacy in Phase I trials (Malkin et al., 2005) and recently has been tested in Phase II clinical trial (Sagara et al., 2009). The essential role of PfAMA-1 in parasite survival and high level of immunogenicity during natural infection in human makes this protein an attractive candidate for gene diversity analysis.
AMA-1 is an 83-kDa type I integral membrane protein with an ectodomain organized in three domains (domain I-III) (Howell et al., 2003, Healer et al., 2005). It is synthesized late during the development of parasite schizonts and processed to a 66-kDa form that relocates to the surfaces of mature merozoites (Bannister et al., 2003, Howell et al., 2001). The domain-I of PfAMA-1 is the most diverse region of this antigen (Escalante et al., 2001, Marshall et al., 1996, Polley and Conway, 2001) and appears to be a major target of anti-AMA-1 protective antibodies (Dutta et al., 2003, Kocken et al., 2002, Mardani et al., 2012). To this end, the present study examines the level of genetic diversity of domain-I of P. falciparum ama-1 gene from Kolkata, West Bengal, and compares the diversity parameters between different regions of India by collating this data with those available in GenBank. We also predict the 3D structure and the protein–protein dockings of mutant PfAMA-1 using different in silico strategies to evaluate the impact of amino acid substitutions on the binding affinities of AMA-1 with its functional partners.
Section snippets
Study site and sample collection
The study was conducted in Kolkata, West Bengal from India. Malaria transmission is seasonal (from May to July and then again September–November) in Kolkata with a predominance of P. falciparum infection and the proportion is in the range of 50–70 (Joshi et al., 2007). Peripheral blood (2–3 ml) was drawn from mild symptomatic malaria patients from the malaria clinic attached to The Calcutta School of Tropical Medicine, Kolkata, India between October 2008 and November 2009. The study protocol for
Sequence diversity and population structure of Kolkata clinical isolates
The main goal of this study was to analyze the genetic diversity of P. falciparum field isolates from Kolkata, West Bengal based on the hypervariable domain-I of ama-1 gene sequences and to disentangle the footprints of natural selection and demographic forces responsible for maintaining the observed diversity. The assessment of a 449 bp sequence spanning 468–917 bp region of the ama-1 gene in 100 P. falciparum clinical isolates from Kolkata (NCBI GenBank accession no: KC476551-KC476650) resulted
Discussion
In the present study, we have combined a comparative evolutionary analysis of ama-1 domain-I gene sequence, with the docking approaches to infer protein–protein interactions between AMA-1 and RON2 and IgNAR to dissect the relative impact of recombination, selection and demographic history on the observed sequence diversity of the Kolkata P. falciparum parasite population. An analysis of a 449 bp sequence of Pfama-1 gene in 100 clinical isolates of Kolkata revealed very high haplotype diversity.
Acknowledgements
The authors are grateful to all study participants for their cooperation. This work has been supported by the funding from Department of Science and Technology, New Delhi (SERC Fast Track Scheme: SR/FTP/L-56/2005 dated 25.04.2006) and University Grants Commission (Major Research Project-F. No 33-232/2007(SR) dated 13.03.2008). We also thank CAS (UGC), DST-FIST, and IPLS (DBT) for providing some of the instrument facilities at the Department of Biochemistry, University of Calcutta. MB is
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