Genetic Diversity and Natural Selection of the Plasmodium knowlesi Circumsporozoite Protein Nonrepeat Regions

BACKGROUND: Plasmodium knowlesi is a simian malaria parasite that has been identified to cause malaria in humans. To date, several thousand cases of human knowlesi malaria have been reported around Southeast Asia. Thus far, there is no detailed study on genetic diversity and natural selection of P. knowlesi circumsporozoite protein (CSP), a prominent surface antigen on the sporozoite of the parasite. In the present study, the genetic diversity and natural selection acting on the nonrepeat regions of the gene encoding P. knowlesi CSP were investigated, focusing on the T-cell epitope regions at the C-terminal of the protein. METHODS: Blood samples from 32 knowlesi malaria patients and 2 wild monkeys (Macaca fascicularis) were used. The CSP of the P. knowlesi isolates was amplified by PCR, cloned into Escherichia coli, and sequenced. The nonrepeat regions of the CSP gene were analysed for genetic diversity, natural selection and haplotypic grouping using MEGA5 and DnaSP version 5.10.00 programmes. A haplotype network was constructed based on the C-terminal (Th2R/Th3R) T-cell epitope regions using the Median-Joining method in the NETWORK version 4.6.1.2 programme. Previously published sequences from other regions (Malaysia Borneo, Singapore) were also included in the analysis. RESULTS: A total of 123 P. knowlesi CSP sequences were analysed. Multiple sequence alignment revealed 58 amino acid changes, and 42 novel amino acid haplotypes were identified. Polymorphism was higher in the C-terminal Th2R/Th3R epitope (π = 0.0293, n = 123) region compared to the overall combined nonrepeat regions (π = 0.0120, n = 123). Negative natural selection was observed within the nonrepeat regions of the CSP gene. Within the C-terminal Th2R/Th3R epitope regions, there was evidence of slight positive selection. Based on haplotype network analysis of the Th2R/Th3R regions, five abundant haplotypes were identified. Sharing of haplotypes between humans and macaques were observed. CONCLUSION: This study contributes to the understanding of the type and distribution of naturally occurring polymorphism in the P. knowlesi CSP gene. This study also provides a measurement of the genetic diversity of P. knowlesi and identifies the predominant haplotypes within Malaysia based on the C-terminal Th2R/Th3R regions.