Jeremy Ryan, De Silva (2017) Genetic diversity study, expression and immunocharacterization of Plasmodium knowlesi merozoite surface protein-3 (MSP-3) in Escherichia /Jeremy Ryan De Silva. PhD thesis, University of Malaya.
Abstract
Malaria is an infectious disease that causes considerable mortality and morbidity globally each year. The Merozoite Surface Protein-3 (MSP-3) is a multigene family of proteins that is found on the surface of the Plasmodium merozoite. Multiple paralogs of the gene can be found in Plasmodium parasites and orthologs of this gene have also been identified in many different species of Plasmodium including P. knowlesi. In this study, 23 P. knowlesi clinical isolates were studied to evaluate genetic diversity, polymorphisms and natural selection acting on the P. knowlesi MSP-3 (pkmsp3) gene. The pkmsp3 gene which contains a signal peptide, an alanine rich central domain denoted Domain A, and a C-terminal region denoted Domain B was amplified by PCR, cloned into Escherichia coli and sequenced. A total of 48 pkmsp3 sequences were obtained. The nucleotide diversity (π) of the full length sequence was found to be marginally higher relative to other P. knowlesi functional genes. Diversity was found to be higher for Domain A (π: 0.035 ± 0.012) and lower in Domain B (π: 0.028 ± 0.002). Comparisons and analysis with P. knowlesi strain H as a reference sequence showed mutations at 339 positions and these amino acid sequences could be categorised into 42 haplotypes. Analysis of the phylogenetic tree and haplotype network revealed that the haplotypes clustered and split into two main distinct groups. The Tajima’s D, Fu & Li’s D* and F* tests and codon based Z-test showed no significant departure from neutrality however, estimations of the dN/dS ratio for Domain B was 0.6, indicating that this particular domain may be under purifying selection. The pkmsp3 gene was then expressed as a ~34 kDa recombinant protein pkMSP- 3 using an E. coli expression system. The sensitivity and specificity of the purified proteins were evaluated in Western Blot and ELISA. In Western Blot, pkMSP-3 exhibited a sensitivity of 61.0% and a specificity of 100.0%. In ELISA, the pkMSP-3 protein was iv found to have a sensitivity of 100.0% and a specificity of 97.1%. High sensitivity in ELISA and high specificity in Western Blot indicates that this protein holds potential as an immunodiagnostic marker if both assays are used in tandem for diagnosis. This study then further aimed to evaluate the immunogenicity of pkMSP-3 using a mouse model to evaluate if it had any potential to inhibit P. knowlesi merozoite invasion into human normocytes. Mice were immunized with pkMSP-3 and displayed significantly higher levels of the cytokine interferon-gamma, interleukin-2 and interleukin-6 when compared to cytokine levels in negative control mice. The pkMSP-3 raised antibodies were found to have a high endpoint titre with IgG1 having the highest isotype distribution followed by IgG2b, IgG2a, IgG3 and finally IgG2c. The localization of pkMSP-3-immunized mice antibodies was studied by immunofluorescence microscopy where the antibodies were found to localize around the membrane of individual merozoites. Lastly, a merozoite invasion assay was carried out using human normocytes treated with or without pkMSP-3 monoclonal antibody. Human normocytes treated with pkMSP-3 monoclonal antibodies had a percent inhibition of 49.6% where invasion rates were almost halved compared to untreated human normocytes.
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